A new human species of aldolase A mRNA from fibroblasts

A full-length cDNA aldolase A clone was isolated from a human fibroblast cDNA library and completely sequenced. Excluding the poly(A) tail, the clone covers 1095 base pairs (bp) of the coding region, plus 199 bp downstream for the termination codon and 146 bp upstream for the initiation codon, within a total of 1440 bp. Primer extension experiments performed with human cultured fibroblast mRNA indicate an elongated product of a further 40 bp. These results evaluated together with those obtained in a concurrent study concerning aldolase A mRNA isolated from human liver are direct evidence of aldolase A mRNA multiplicity in man. The data also suggest the existence in mammals of three different classes of aldolase A mRNA, which would account for tissue specificity and resurgence of foetal expression in tumors.     

A novel class of ammonium assimilation mutants of the photosynthetic bacterium Rhodobacter capsulatus

Nitrogen assimilation in Rhodobacter capsulatus has been shown to proceed via the coupled action of glutamine synthetase (GS) and glutamate synthase (GOGAT) with no measurable glutamate dehydrogenase (GDH) present. We have recently isolated a novel class of mutants of R. capsulatus strain B100 that lacks a detectable GOGAT activity but is able to grow at wild type rates under nitrogen-fixing conditions. While NH ₄ ⁺ -supported growth in the mutants was normal under anaerobic/photosynthetic conditions, the growth rate was decreased under aerobic conditions. Ammonium and methylammonium uptake experiments indicated that there was a clear difference in the ammonium assimilatory capabilities in these mutants under aerobic versus anaerobic growth. Regulation of expression of a nifH : : lacZ fusion in these mutants was not impaired. The possible existence of alternative ammonium assimilatory pathways is discussed.     

Evaluation of nucleolar organizer region-associated proteins in endometrial pathology.

In the current study the argyrophil staining technique for NOR proteins (Ag-NORs) has been performed on cases of different endometrial lesions, trying to find an aid in differentiating atypical hyperplasia from well differentiated carcinoma in biopsy specimens. We conclude that the Ag-NOR count, even though in endometrial carcinoma is significantly exceeding that of atypical hyperplastic endometrium, could be a misleading discriminator, because of a wide overlap of values in individual cases.     

The lepidopteran mitochondrial control region: structure and evolution

For several species of lepidoptera, most of the approximately 350-bp mitochondrial control-region sequences were determined. Six of these species are in one genus, Jalmenus; are closely related; and are believed to have undergone recent rapid speciation. Recent speciation was supported by the observation of low interspecific sequence divergence. Thus, no useful phylogeny could be constructed for the genus. Despite a surprising conservation of control-region length, there was little conservation of primary sequences either among the three lepidopteran genera or between lepidoptera and Drosophila. Analysis of secondary structure indicated only one possible feature in common--inferred stem loops with higher-than-random folding energies-- although the positions of the structures in different species were unrelated to regions of primary sequence similarity. We suggest that the conserved, short length of control regions is related to the observed lack of heteroplasmy in lepidopteran mitochondrial genomes. In addition, determination of flanking sequences for one Jalmenus species indicated (i) only weak support for the available model of insect 12S rRNA structure and (ii) that tRNA translocation is a frequent event in the evolution of insect mitochondrial genomes.      

Glutamine repeats and inherited neurodegenerative diseases: molecular aspects

Several dominantly inherited, late onset, neurodegenerative diseases are due to expansion of CAG repeats, leading to expansion of glutamine repeats in the affected proteins. These proteins are of very different sizes and, with one exception, show no sequence homology to known proteins or to each other; their functions are unknown. In some, the glutamine repeat starts near the N-terminus, in another near the middle and in another near the C-terminus, but regardless of these differences, no disease has been observed in individuals with fewer than 37 repeats, and absence of disease has never been found in those with more than 41 repeats. Protein constructs with more than 41 repeats are toxic to E. coli and to CHO cells in culture, and they elicit ataxia in transgenic mice. These observations argue in favour of a distinct change of structure associated with elongation beyond 37–41 glutamine repeats. The review describes experiments designed to find out what these structures might be and how they could influence the properties of the proteins of which they form part. Poly- -glutamines form pleated sheets of β-strands held together by hydrogen bonds between their amides. Incorporation of glutamine repeats into a small protein of known structure made it associate irreversibly into oligomers. That association took place during the folding of the protein molecules and led to their becoming firmly interlocked by either strand- or domain-swapping. Thermodynamic considerations suggest that elongation of glutamine repeats beyond a certain length may lead to a phase change from random coils to hydrogen-bonded hairpins. Possible mechanisms of expansion of CAG repeats are discussed in the light of looped DNA model structures.     

Development of cell surface saccharides on embryonic pancreatic cells

Using a battery of seven lectin-ferritin conjugates as probes for cell surface glycoconjugates, we have studied the pattern of plasmalemmal differentiation of cells in the embryonic rat pancreas from day 15 in utero to the early postpartum stage. Our results indicate that differentiation of plasmalemmal glycoconjugates on acinar, endocrine, and centroacinar cells is temporally correlated with development and is unique for each cell type, as indicated by lectin-ferritin binding. Specifically, (a) expression of adult cell surface saccharide phenotype can be detected on presumptive acinar cells as early as 15 d in utero, as indicated by soybean agglutinin binding, and precedes development of intracellular organelles characteristic of mature acinar cells; (b) maturation of the plasmalemma of acinar cells is reached after intracellular cytodifferentiation is completed, as indicated by appearance of Con A and fucoselectin binding sites only at day 19 of development; conversely, maturation of the endocrine cell plasmalemma is accompanied by "loss" (masking) of ricinus communis II agglutinin receptors; and (c) binding sites for fucose lectins and for soybean agglutinin are absent on endocrine and centroacinar cells at all stages examined. We conclude that acinar, centroacinar, and endocrine cells develop from a common progenitor cell(s) whose plasmalemmal carbohydrate composition resembles most closely that of the adult centroacinar cell. Finally, appearance of acinar lumina beginning at approximately 17 d in utero is accompanied by differenetiation of apical and basolateral plasmalemmal domains of epithelial cells, as indicated by enhanced binding of several lectin-ferritin conjugates to the apical plasmalemmal, a pattern that persists from this stage through adult life.     

Cellular Resistance Against Troxacitabine in Human Cell Lines and Pediatric Patient Acute Myeloid Leukemia Blast Cells

Troxacitabine is a cytotoxic deoxycytidine analogue with an unnatural L-configuration, which is activated by deoxycytidine kinase (dCK). The configuration is responsible for differences in the uptake and metabolism of troxacitabine compared to other deoxynucleoside analogues. To determine whether troxacitabine has an advantage over other nucleoside analogues several cell lines resistant to cladribine and gemcitabine were exposed to troxacitabine, while blast cells from pediatric leukemia patients were tested for cross-resistance with other deoxynucleoside analogues. The gemcitabine resistant AG6000 (IC₅₀: >3000 nM), and the cladribine resistant CEM (IC₅₀: 150 nM) and HL-60 (IC₅₀: >3000 nM) cell lines, all with no or decreased dCK expression, were less sensitive to troxacitabine than their wild type counterparts (IC₅₀; A2780: 410, CEM: 71 and HL-60: 158 nM). dCK protein expression in CEM was higher than in HL-60, which, in turn, was higher than in A2780. Catalytically inactive p53 seems to increase the sensitivity to troxacitabine. The patient samples showed a large range of sensitivity to troxacitabine, similar to other deoxynucleoside analogues. Cross-resistance with all other deoxynucleoside analogues was observed.     

Knockout of pgdS and ggt genes improves γ‐PGA yield in B. subtilis

One of the emerging biopolymers that are currently under active investigation is bacterial poly(γ‐glutamic acid) (γ‐PGA). However, before its full industrial exploitation, a substantial increase in microbial productivity is required. γ‐PGA obtained from the Bacillus subtilis laboratory strain 168 offers the advantage of a producer characterized by a well defined genetic framework and simple manipulation techniques. In this strain, the knockout of genes for the major γ‐PGA degrading enzymes, pgdS and ggt, leads to a considerable improvement in polymer yield, which attains levels analogous to the top wild γ‐PGA producer strains. This study highlights the convenience of using the laboratory strain of B. subtilis over wild isolates in designing strain improvement strategies aimed at increasing γ‐PGA productivity. Biotechnol. Bioeng. 2013; 110: 2006–2012. © 2013 Wiley Periodicals, Inc.