Minisatellite marker analysis of Trypanosoma brucei: Reconciliation of clonal, panmictic, and epidemic population genetic structures

The African trypanosome, Trypanosoma brucei, has been shown to undergo genetic exchange in the laboratory, but controversy exists as to the role of genetic exchange in natural populations. Much of the analysis to date has been derived from isoenzyme or randomly amplified polymorphic DNA data with parasite material from a range of hosts and geographical locations. These markers fail to distinguish between the human infective (T. b. rhodesiense) and nonhuman infective (T. b. brucei) “subspecies” so that parasites derived from hosts other than humans potentially contain both subspecies. To overcome some of the inherent problems with the use of such markers and diverse populations, we have analyzed a well-defined population from a discrete geographical location (Busoga, Uganda) using three recently described minisatellite markers. The parasites were primarily isolated from humans and cattle with the latter isolates further characterized by their ability to resist lysis by human serum (equivalent to human infectivity). The minisatellite markers show high levels of polymorphism, and from the data obtained we conclude that T. b. rhodesiense is genetically isolated from T. b. brucei and can be unambiguously identified by its multilocus genotype. Analysis of the genotype frequencies in the separated T. b. brucei and T. b. rhodesiense populations shows the former has an epidemic population structure whereas the latter is clonal. This finding suggests that the strong linkage disequilibrium observed in previous analyses, where human and nonhuman infective trypanosomes were not distinguished, results from the treatment of two genetically isolated populations as a single population.

A bait and switch hapten strategy generates catalytic antibodies for phosphodiester hydrolysis

General base catalysis supplied by the histidine-12 (H-12) residue of ribonuclease (RNase) A has long been appreciated as a major component of the catalytic power of the enzyme. In an attempt to harness the catalytic power of a general base into antibody catalysis of phosphodiester bond hydrolysis, the quaternary ammonium phosphate 1 was used as a bait and switch hapten. Based on precedence, it was rationalized that this positively charged hapten could induce a counter-charged residue in the antibody binding site at a locus suitable for it to deprotonate the 2′-hydroxyl group of the anhydroribitol phosphodiester substrate 2. After murine immunization with hapten 1, mAb production yielded a library of 35 antibodies that bound to a BSA-1 conjugate. From this panel, two were found to catalyze the cyclization-cleavage of phosphodiester 2. Kinetic studies at pH 7.49 (Hepes, 20 mM) and 25°C showed that the most active antibody, MATT.F-1, obeyed classical Michaelis–Menten kinetics with a Km = 104 μM, a kcat = 0.44 min−1, and a kcat/kuncat = 1.7 × 103. Hapten 1 stoichiometrically inhibits the catalytic activity of the antibody. MATT.F-1 is the most proficient antibody–catalyst (1.6 × 107 M−1) yet generated for the function of phosphodiester hydrolysis and emphasizes the utility of the bait and switch hapten paradigm when generating antibody catalysts for processes for which general-base catalysis can be exploited.

Gel catalysts that switch on and off

We report development of a polymer gel with a catalytic activity that can be switched on and off when the solvent composition is changed. The gel consists of two species of monomers. The major component, N-isopropylacrylamide, makes the gel swell and shrink in response to a change in composition of ethanol/water mixtures. The minor component, vinylimidazole, which is capable of catalysis, is copolymerized into the gel network. The reaction rate for catalytic hydrolysis of p-nitrophenyl caprylate was small when the gel was swollen. In contrast, when the gel was shrunken, the reaction rate increased 5 times. The activity changes discontinuously as a function of solvent composition, thus the catalysis can be switched on and off by an infinitesimal change in solvent composition. The kinetics of catalysis by the gel in the shrunken state is well described by the Michaelis–Menten formula, indicating that the absorption of the substrate by the hydrophobic environment created by the N-isopropylacrylamide polymer in the shrunken gel is responsible for enhancement of catalytic activity. In the swollen state, the rate vs. active site concentration is linear, indicating that the substrate absorption is not a primary factor determining the kinetics. Catalytic activity of the gel is studied for substrates with various alkyl chain lengths; of those studied the switching effect is most pronounced for p-nitrophenyl caprylate.

A trans-acting RNA as a control switch in Escherichia coli: DsrA modulates function by forming alternative structures

DsrA is an 87-nucleotide regulatory RNA of Escherichia coli that acts in trans by RNA–RNA interactions with two different mRNAs, hns and rpoS. DsrA has opposite effects on these transcriptional regulators. H-NS levels decrease, whereas RpoS (σs) levels increase. Here we show that DsrA enhances hns mRNA turnover yet stabilizes rpoS mRNA, either directly or via effects on translation. Computational and RNA footprinting approaches led to a refined structure for DsrA, and a model in which DsrA interacts with the hns mRNA start and stop codon regions to form a coaxial stack. Analogous bipartite interactions exist in eukaryotes, albeit with different regulatory consequences. In contrast, DsrA base pairs in discrete fashion with the rpoS RNA translational operator. Thus, different structural configurations for DsrA lead to opposite regulatory consequences for target RNAs.

Tight Asp-85–Thr-89 association during the pump switch of bacteriorhodopsin

Unidirectional proton transport in bacteriorhodopsin is enforced by the switching machinery of the active site. Threonine 89 is located in this region, with its O—H group forming a hydrogen bond with Asp-85, the acceptor for proton transfer from the Schiff base of the retinal chromophore. Previous IR spectroscopy of [3-18O]threonine-labeled bacteriorhodopsin showed that the hydrogen bond of the O—D group of Thr-89 in D2O is strengthened in the K photocycle intermediate. Here, we show that the strength and orientation of this hydrogen bond remains unchanged in the L intermediate and through the M intermediate. Furthermore, a strong interaction between Asp-85 and the O—H (O—D) group of Thr-89 in M is indicated by a shift in the C⩵O stretching vibration of the former because of 18O substitution in the latter. Thus, the strong hydrogen bond between Asp-85 and Thr-89 in K persists through M, contrary to structural models based on x-ray crystallography of the photocycle intermediates. We propose that, upon photoisomerization of the chromophore, Thr-89 forms a tight, persistent complex with one of the side-chain oxygens of Asp-85 and is thereby precluded from participating in the switching process. On the other hand, the loss of hydrogen bonding at the other oxygen of Asp-85 in M may be related to the switching event.

Calmodulin kinase is a molecular switch for cardiac excitation –contraction coupling

Signaling between cell membrane-bound L-type Ca2+ channels (LTCC) and ryanodine receptor Ca2+ release channels (RyR) on sarcoplasmic reticulum (SR) stores grades excitation–contraction coupling (ECC) in striated muscle. A physical connection regulates LTCC and RyR in skeletal muscle, but the molecular mechanism for coordinating LTCC and RyR in cardiomyocytes, where this physical link is absent, is unknown. Calmodulin kinase (CaMK) has characteristics suitable for an ECC coordinating molecule: it is activated by Ca2+/calmodulin, it regulates LTCC and RyR, and it is enriched in the vicinity of LTCC and RyR. Intact cardiomyocytes were studied under conditions where CaMK activity could be controlled independently of intracellular Ca2+ by using an engineered Ca2+-independent form of CaMK and a highly specific CaMK inhibitory peptide. CaMK reciprocally enhanced L-type Ca2+ current and reduced release of Ca2+ from the SR while increasing SR Ca2+ content. These findings support the hypothesis that CaMK is required to functionally couple LTCC and RyR during cardiac ECC.

Von Willebrand factor propeptide as a marker of disease activity in systemic sclerosis (scleroderma)

In 44 consecutive patients with systemic sclerosis (SSc), plasma concentrations of von Willebrand factor (vWf) were higher than those of the vWf propeptide, but the propeptide showed less variability within patient subgroups. Higher values of the propeptide were observed in patients with early pulmonary involvement. A closer correlation of the propeptide than of vWf to biochemical markers of activity was also evident. Our results suggest that the propeptide, despite a shorter circulating half-time and lower plasma concentrations than vWf, is more useful in the assessment of disease activity in SSc.

Switch from Myc/Max to Mad1/Max binding and decrease in histone acetylation at the telomerase reverse transcriptase promoter during differentiation of HL60 cells

Recent evidence suggests that the Myc and Mad1 proteins are implicated in the regulation of the gene encoding the human telomerase reverse transcriptase (hTERT), the catalytic subunit of telomerase. We have analyzed the in vivo interaction between endogenous c-Myc and Mad1 proteins and the hTERT promoter in HL60 cells with the use of the chromatin immunoprecipitation assay. The E-boxes at the hTERT proximal promoter were occupied in vivo by c-Myc in exponentially proliferating HL60 cells but not in cells induced to differentiate by DMSO. In contrast, Mad1 protein was induced and bound to the hTERT promoter in differentiated HL60 cells. Concomitantly, the acetylation of the histones at the promoter was significantly reduced. These data suggest that the reciprocal E-box occupancy by c-Myc and Mad1 is responsible for activation and repression of the hTERT gene in proliferating and differentiated HL60 cells, respectively. Furthermore, the histone deacetylase inhibitor trichostatin A inhibited deacetylation of histones at the hTERT promoter and attenuated the repression of hTERT transcription during HL60 cell differentiation. In addition, trichostatin A treatment activated hTERT transcription in resting human lymphocytes and fibroblasts. Taken together, these results indicate that acetylation/deacetylation of histones is operative in the regulation of hTERT expression.

A splicing switch and gain-of-function mutation in FgfR2-IIIc hemizygotes causes Apert/Pfeiffer-syndrome-like phenotypes

Intercellular signaling by fibroblast growth factors plays vital roles during embryogenesis. Mice deficient for fibroblast growth factor receptors (FgfRs) show abnormalities in early gastrulation and implantation, disruptions in epithelial–mesenchymal interactions, as well as profound defects in membranous and endochondrial bone formation. Activating FGFR mutations are the underlying cause of several craniosynostoses and dwarfism syndromes in humans. Here we show that a heterozygotic abrogation of FgfR2-exon 9 (IIIc) in mice causes a splicing switch, resulting in a gain-of-function mutation. The consequences are neonatal growth retardation and death, coronal synostosis, ocular proptosis, precocious sternal fusion, and abnormalities in secondary branching in several organs that undergo branching morphogenesis. This phenotype has strong parallels to some Apert's and Pfeiffer's syndrome patients.

Allostatic load as a marker of cumulative biological risk: MacArthur studies of successful aging

Allostatic load (AL) has been proposed as a new conceptualization of cumulative biological burden exacted on the body through attempts to adapt to life's demands. Using a multisystem summary measure of AL, we evaluated its capacity to predict four categories of health outcomes, 7 years after a baseline survey of 1,189 men and women age 70–79. Higher baseline AL scores were associated with significantly increased risk for 7-year mortality as well as declines in cognitive and physical functioning and were marginally associated with incident cardiovascular disease events, independent of standard socio-demographic characteristics and baseline health status. The summary AL measure was based on 10 parameters of biological functioning, four of which are primary mediators in the cascade from perceived challenges to downstream health outcomes. Six of the components are secondary mediators reflecting primarily components of the metabolic syndrome (syndrome X). AL was a better predictor of mortality and decline in physical functioning than either the syndrome X or primary mediator components alone. The findings support the concept of AL as a measure of cumulative biological burden.

Blue Light and Abscisic Acid Independently Induce Heterophyllous Switch in Marsilea quadrifolia1

In natural habitats Marsilea quadrifolia L. produces different types of leaves above and below the water level. In aseptic cultures growth conditions can be manipulated so that leaves of the submerged type are produced continuously. Under such conditions the application of either blue light or an optimal concentration of abscisic acid (ABA) induced the development of aerial-type leaves. When fluridone, an inhibitor of ABA biosynthesis, was added to the culture medium it did not prevent blue light induction of aerial leaf development. During blue light treatment the endogenous ABA level in M. quadrifolia leaves remained unchanged. However, after the plants were transferred to an enriched medium, the ABA level gradually increased, corresponding to a transition in development from the submerged type of leaves to aerial leaves. These results indicate that the blue light signal is not mediated by ABA. Therefore, in the regulation of heterophyllous determination, discrete pathways exist in response to environmental signals.

Dynamic properties of the Ras switch I region and its importance for binding to effectors

We have investigated the dynamic properties of the switch I region of the GTP-binding protein Ras by using mutants of Thr-35, an invariant residue necessary for the switch function. Here we show that these mutants, previously used as partial loss-of-function mutations in cell-based assays, have a reduced affinity to Ras effector proteins without Thr-35 being involved in any interaction. The structure of Ras(T35S)⋅GppNHp was determined by x-ray crystallography. Whereas the overall structure is very similar to wildtype, residues from switch I are completely invisible, indicating that the effector loop region is highly mobile. 31P-NMR data had indicated an equilibrium between two rapidly interconverting conformations, one of which (state 2) corresponds to the structure found in the complex with the effectors. 31P-NMR spectra of Ras mutants (T35S) and (T35A) in the GppNHp form show that the equilibrium is shifted such that they occur predominantly in the nonbinding conformation (state 1). On addition of Ras effectors, Ras(T35S) but not Ras(T35A) shift to positions corresponding to the binding conformation. The structural data were correlated with kinetic experiments that show two-step binding reaction of wild-type and (T35S)Ras with effectors requires the existence of a rate-limiting isomerization step, which is not observed with T35A. The results indicate that minor changes in the switch region, such as removing the side chain methyl group of Thr-35, drastically affect dynamic behavior and, in turn, interaction with effectors. The dynamics of the switch I region appear to be responsible for the conservation of this threonine residue in GTP-binding proteins.

Factors regulating template switch in vitro by viral RNA-dependent RNA polymerases: Implications for RNA–RNA recombination

Copy-choice RNA recombination occurs during viral RNA synthesis when the viral transcription complex switches templates. We demonstrate that RNA-dependent RNA polymerase from bovine viral diarrhea virus and the replicases from three plant-infecting RNA viruses can produce easily detectable recombination products in vitro by switching templates during elongative RNA synthesis. Template sequence and/or structure, and NTP availability affected the frequency of template switch by the transcription complex. Our results provide biochemical support for copy-choice recombination and establish assays for mechanistic analyses of intermolecular RNA recombination in vitro.

In vivo detection of gene expression in liver by 31P nuclear magnetic resonance spectroscopy employing creatine kinase as a marker gene

In vivo assessment of gene expression is desirable to obtain information on the extent and duration of transduction of tissue after gene delivery. We have developed an in vivo, potentially noninvasive, method for detecting virally mediated gene transfer to the liver. The method employs an adenoviral vector carrying the gene for the brain isozyme of murine creatine kinase (CK-B), an ATP-buffering enzyme expressed mainly in muscle and brain but absent from liver, kidney, and pancreas. Gene expression was monitored by 31P magnetic resonance spectroscopy (MRS) using the product of the CK enzymatic reaction, phosphocreatine, as an indicator of transfection. The vector was administered into nude mice by tail vein injection, and exogenous creatine was administered in the drinking water and by i.p. injection of 2% creatine solution before 31P MRS examination, which was performed on surgically exposed livers. A phosphocreatine resonance was detected in livers of mice injected with the vector and was absent from livers of control animals. CK expression was confirmed in the injected animals by Western blot analysis, enzymatic assays, and immunofluorescence measurements. We conclude that the syngeneic enzyme CK can be used as a marker gene for in vivo monitoring of gene expression after virally mediated gene transfer to the liver.

Src, a molecular switch governing gain control of synaptic transmission mediated by N-methyl-d-aspartate receptors

The N-methyl-d-aspartate (NMDA) receptor is a principal subtype of glutamate receptor mediating fast excitatory transmission at synapses in the dorsal horn of the spinal cord and other regions of the central nervous system. NMDA receptors are crucial for the lasting enhancement of synaptic transmission that occurs both physiologically and in pathological conditions such as chronic pain. Over the past several years, evidence has accumulated indicating that the activity of NMDA receptors is regulated by the protein tyrosine kinase, Src. Recently it has been discovered that, by means of up-regulating NMDA receptor function, activation of Src mediates the induction of the lasting enhancement of excitatory transmission known as long-term potentiation in the CA1 region of the hippocampus. Also, Src has been found to amplify the up-regulation of NMDA receptor function that is produced by raising the intracellular concentration of sodium. Sodium concentration increases in neuronal dendrites during high levels of firing activity, which is precisely when Src becomes activated. Therefore, we propose that the boost in NMDA receptor function produced by the coincidence of activating Src and raising intracellular sodium may be important in physiological and pathophysiological enhancement of excitatory transmission in the dorsal horn of the spinal cord and elsewhere in the central nervous system.