Protein kinase D1 inhibition interferes with mitosis progression

Protein kinase D1 (PKD1) plays a vital role in signal transduction, cell proliferation, membrane trafficking, and cancer; however, the majority of the studies up to date had centered primarily on PKD1 functions in interphase, very little is known about its role during cell division. We previously demonstrated that during mitosis PKD1 is activated and associated with centrosomes, spindles, and midbodies. However, these observations did not address whether PKD1 was associated with mitosis regulation. Accordingly, we used rapidly acting PKD-specific inhibitors to examine the contribution of PKD1 the sequence of events in mitosis. We found that although PKD1 overexpression did not affect mitosis progression, suppression of its catalytic activity by two structurally unrelated inhibitors (kb NB 142-70 and CRT 0066101) induced a significant delay in metaphase to anaphase transition time. PKD1 inhibition during mitosis also produced the appearance of abnormal spindles, defects in chromosome alignment, and segregation as well as apoptosis. Thus, these observations indicate that PKD1 activity is associated with mitosis regulation.     

Tyrosine phosphorylation signaling regulates Ca2+ entry by affecting intracellular pH during human sperm capacitation

Capacitation is a mandatory process for the acquisition of mammalian sperm fertilization competence and involves the activation of a complex and still not fully understood system of signaling pathways. Under in vitro conditions, there is an increase in both protein tyrosine phosphorylation (pTyr) and intracellular Ca²⁺ levels in several species. In human sperm, results from our group revealed that pTyr signaling can be blocked by inhibiting proline-rich tyrosine kinase 2 (PYK2). Based on the role of PYK2 in other cell types, we investigated whether the PYK2-dependent pTyr cascade serves as a sensor for Ca ²⁺ signaling during human sperm capacitation. Flow cytometry studies showed that exposure of sperm to the PYK2 inhibitor N-[2-[[[2-[(2,3-dihydro-2-oxo-1 H-indol-5-yl)amino]-5-(trifluoromethyl)-4-pyrimidinyl]amino]methyl]phenyl]- N-methyl-methanesulfonamide hydrate (PF431396) produced a significant and concentration-dependent reduction in intracellular Ca ²⁺ levels during capacitation. Further studies revealed that PF431396-treated sperm exhibited a decrease in the activity of CatSper, a key sperm Ca ²⁺ channel. In addition, time course studies during capacitation in the presence of PF431396 showed a significant and sustained decrease in both intracellular Ca ²⁺ and pH levels after 2 hr of incubation, temporarily coincident with the activation of PYK2 during capacitation. Interestingly, decreases in Ca ²⁺ levels and progressive motility caused by PF431396 were reverted by inducing intracellular alkalinization with NH ₄Cl, without affecting the pTyr blockage. Altogether, these observations support pTyr as an intracellular sensor for Ca ²⁺ entry in human sperm through regulation of cytoplasmic pH. These results contribute to a better understanding of the modulation of the polymodal CatSper and signaling pathways involved in human sperm capacitation.     

Improving bioreactor production of a recombinant glycoprotein in Escherichia coli: Effect of specific growth rate on protein glycosylation and specific productivity

In the last decades bacterial glycoengineering emerged as a new field as the result of the ability to transfer the Campylobacter jejuni N- glycosylation machinery into Escherichia coli for the production of recombinant glycoproteins that can be used as antigens for diagnosis, vaccines, and therapeutics. However, the identification of critical parameters implicated in the production process and its optimization to jump to a productive scale is still required. In this study, we developed a dual expression glycosylation vector for the production of the recombinant glycoprotein AcrA-O157, a novel antigen that allows the serodiagnosis of the infection with enterohemorrhagic E. coli O157 in humans. Volumetric productivity was studied in different culture media and found that 2xYP had 6.9-fold higher productivity than the extensively used LB. Subsequently, bioreactor batch and exponential-fed-batch cultures were designed to determine the influence of the specific growth rate (μ) on AcrA-O157 glycosylation efficiency, production kinetics, and specific productivity. At μ_max, AcrA glycosylation with O157-polysaccharide and the specific synthesis rate were maximal, constituting the optimal physiological condition for AcrA-O157 production. Our findings should be considered for the design, optimization, and scaling up of AcrA-O157 production and other recombinant glycoproteins attractive for industrial applications.     

Detection of intracellular iron by its regulatory effect

Intracellular iron regulates gene expression by inhibiting the interaction of iron regulatory proteins (IRPs) with RNA motifs called iron-responsive elements (IREs). To assay this interaction in living cells we have developed two fluorescent IRE-based reporters that rapidly, reversibly, and specifically respond to changes in cellular iron status as well as signaling that modifies IRP activity. The reporters were also sufficiently sensitive to distinguish apo- from holotransferrin in the medium, to detect the effect of modifiers of the transferrin pathway such as HFE, and to detect the donation or chelation of iron by siderophores bound to the lipocalin neutrophil gelatinase-associated lipocalin (Ngal). In addition, alternative configurations of the IRE motif either enhanced or repressed fluorescence, permitting a ratio analysis of the iron-dependent response. These characteristics make it possible to visualize iron-IRP-IRE interactions in vivo. iron regulatory proteins; iron-responsive element; labile iron pool; transferrin; HFE; neutrophil gelatinase-associated lipocalin; siderophore     

Photo-CIDNP 13C magic angle spinning NMR on bacterial reaction centres: exploring the electronic structure of the special pair and its surroundings

Photochemically induced dynamic nuclear polarisation (photo-CIDNP) in intact bacterial reaction centres has been observed by 13C-solid state NMR under continuous illumination with white light. Strong intensity enhancement of 13C NMR signals of the aromatic rings allows probing the electronic ground state of the two BChl cofactors of the special pair at the molecular scale with atomic selectivity. Differences between the two BChl cofactors are discussed. Several aliphatic 13C atoms of cofactors, as well as 13C atoms of the imidazole ring of histidine residue(s), show nuclear-spin polarisation to the same extent as the aromatic nuclei of the cofactors. Mechanisms and applications of polarisation transfer are discussed.     

Visualizing metabolic activity on a genome-wide scale

MOTIVATION: To enhance the exploration of gene expression data in a metabolic context, one requires an application that allows the integration of this data and which represents this data in a (genome-wide) metabolic map. The layout of this metabolic map must be highly flexible to enable discoveries of biological phenomena. Moreover, it must allow the simultaneous representation of additional information about genes and enzymes. Since the layout and properties of existing maps did not fulfill our requirements, we developed a new way of representing gene expression data in metabolic charts. RESULTS: ViMAc generates user-specified (genome-wide) metabolic maps to explore gene expression data. To enhance the interpretation of these maps information such as sub-cellular localization is included. ViMAc can be used to analyse human or yeast expression data obtained with DNA microarrays or SAGE. We introduce our metabolic map method and demonstrate how it can be applied to explore DNA microarray data for yeast. Availability: ViMAc is freely available for academic institutions on request from the authors.     

B-branch electron transfer in reaction centers of Rhodobacter sphaeroides assessed with site-directed mutagenesis

Mutants of Rhodobacter (Rba.) sphaeroides are described which were designed to study electron transfer along the so-called B-branch of reaction center (RC) cofactors. Combining the mutation L(M214)H, which results in the incorporation of a bacteriochlorophyll, β, for H_A [Kirmaier et al. (1991) Science 251: 922–927] with two mutations, G(M203)D and Y(M210)W, near B_A, we have created a double and a triple mutant with long lifetimes of the excited state P^* of the primary donor P, viz. 80 and 160 ps at room temperature, respectively. The yield of P⁺Q_A ⁻ formation in these mutants is reduced to 50 and 30%, respectively, of that in wildtype RCs. For both mutants, the quantum yield of P⁺H_B ⁻ formation was less than 10%, in contrast to the 15% B-branch electron transfer demonstrated in RCs of a similar mutant of Rba. capsulatus with a P^* lifetime of 15 ps [Heller et al. (1995) Science 269: 940–945]. We conclude that the lifetime of P^* is not a governing factor in switching to B-branch electron transfer. The direct photoreduction of the secondary quinone, Q_B, was studied with a triple mutant combining the G(M203)D, L(M214)H and A(M260)W mutations. In this triple mutant Q_A does not bind to the reaction center [Ridge et al. (1999) Photosynth Res 59: 9–26]. It is shown that B-branch electron transfer leading to P⁺Q_B ⁻ formation occurs to a minor extent at both room temperature and at cryogenic temperatures (about 3% following a saturating laser flash at 20 K). In contrast, in wildtype RCs P⁺Q_B ⁻ formation involves the A-branch and does not occur at all at cryogenic temperatures. Attempts to accumulate the P⁺Q_B ⁻ state under continuous illumination were not successful. Charge recombination of P⁺Q_B ⁻ formed by B-branch electron transfer in the new mutant is much faster (seconds) than has been previously reported for charge recombination of P⁺Q_B ⁻ trapped in wildtype RCs (10⁵ s) [Kleinfeld et al. (1984b) Biochemistry 23: 5780–5786]. This difference is discussed in light of the different binding sites for Q_B and Q_B ⁻ that recently have been found by X-ray crystallography at cryogenic temperatures [Stowell et al. (1997) Science 276: 812–816]. We present the first low-temperature absorption difference spectrum due to P⁺Q_B ⁻. This revised version was published online in June 2006 with corrections to the Cover Date.     

Nucleic acid quantification by delta pH measurement

Nucleic acids are quantitated by UV absorbance measurement, fluorimetry, or hybridization. While the latter method is time-consuming and requires exact knowledge of the sequence, spectroscopic methods require that the sample does not contain UV-absorbing or fluorescent material. An enzymatic method is the measurement of the hyperchromic change upon cleavage of the nucleic acids by nucleases (Kunitz assay). A variation of this assay makes use of the acidification of the solution upon cleavage. We demonstrate here that microgram nucleic acid quantities can be determined when one employs highly active nonspecific nucleases in conjunction with an instrumental setup consisting of a temperature-controlled mixing chamber and miniaturized pH electrodes. Because this method determines the total amount of phosphodiester bonds cleaved, it is independent of the composition or the secondary structure of the nucleic acid and, under certain precautions, represents a simple and robust alternative to optical assays for the determination of either the total nucleic acid concentration or the activity of nucleases in biochemical samples.     

The Evolutionary History of the Genus Acanthamoeba and the Identification of Eight New 18S rRNA Gene Sequence Types

ABSTRACT The 18S rRNA gene ( Rns ) phylogeny of Acanthamoeba is being investigated as a basis for improvements in the nomenclature and taxonomy of the genus. We previously analyzed Rns sequences from 18 isolates from morphological groups 2 and 3 and found that they fell into four distinct evolutionary lineages we called sequence types T1-T4. Here, we analyzed sequences from 53 isolates representing 16 species and including 35 new strains. Eight additional lineages (sequence types T5-T12) were identified. Four of the 12 sequence types included strains from more than one nominal species. Thus, sequence types could be equated with species in some cases or with complexes of closely related species in others. The largest complex, sequence type T4, which contained six closely related nominal species, included 24 of 25 keratitis isolates. Rns sequence variation was insufficient for full phylogenetic resolution of branching orders within this complex, but the mixing of species observed at terminal nodes confirmed that traditional classification of isolates has been inconsistent. One solution to this problem would be to equate sequence types and single species. Alternatively, additional molecular information will be required to reliably differentiate species within the complexes. Three sequence types of morphological group 1 species represented the earliest divergence in the history of the genus and, based on their genetic distinctiveness, are candidates for reclassification as one or more novel genera.