Compression, gain, and nonlinear distortion in an active cochlear model with subpartitions

The propagation of inhomogeneous, weakly nonlinear waves is considered in a cochlear model having two degrees of freedom that represent the transverse motions of the tectorial and basilar membranes within the organ of Corti. It is assumed that nonlinearity arises from the saturation of outer hair cell active force generation. I use multiple scale asymptotics and treat nonlinearity as a correction to a linear hydroelastic wave. The resulting theory is used to explain experimentally observed features of the response of the cochlear partition to a pure tone, including: the amplification of the response in a healthy cochlea vs a dead one; the less than linear growth rate of the response to increasing sound pressure level; and the amount of distortion to be expected at high and low frequencies at basal and apical locations, respectively. I also show that the outer hair cell nonlinearity generates retrograde waves.

The Escherichia coli SOS mutagenesis proteins UmuD and UmuD′ interact physically with the replicative DNA polymerase

The Escherichia coli umuDC operon is induced in response to replication-blocking DNA lesions as part of the SOS response. UmuD protein then undergoes an RecA-facilitated self-cleavage reaction that removes its N-terminal 24 residues to yield UmuD′. UmuD′, UmuC, RecA, and some form of the E. coli replicative DNA polymerase, DNA polymerase III holoenzyme, function in translesion synthesis, the potentially mutagenic process of replication over otherwise blocking lesions. Furthermore, it has been proposed that, before cleavage, UmuD together with UmuC acts as a DNA damage checkpoint system that regulates the rate of DNA synthesis in response to DNA damage, thereby allowing time for accurate repair to take place. Here we provide direct evidence that both uncleaved UmuD and UmuD′ interact physically with the catalytic, proofreading, and processivity subunits of the E. coli replicative polymerase. Consistent with our model proposing that uncleaved UmuD and UmuD′ promote different events, UmuD and UmuD′ interact differently with DNA polymerase III: whereas uncleaved UmuD interacts more strongly with β than it does with α, UmuD′ interacts more strongly with α than it does with β. We propose that the protein–protein interactions we have characterized are part of a higher-order regulatory system of replication fork management that controls when the umuDC gene products can gain access to the replication fork.

Tertiary hypothyroidism and hyperglycemia in mice with targeted disruption of the thyrotropin-releasing hormone gene

Thyrotropin-releasing hormone (TRH) is a brain hypothalamic hormone that regulates thyrotropin (TSH) secretion from the anterior pituitary and is ubiquitously distributed throughout the brain and other tissues including pancreas. To facilitate studies into the role of endogenous TRH, we have used homologous recombination to generate mice that lack TRH. These TRH−/− mice are viable, fertile, and exhibit normal development. However, they showed obvious hypothyroidism with characteristic elevation of serum TSH level and diminished TSH biological activity. Their anterior pituitaries exhibited an apparent decrease in TSH immunopositive cells that was not due to hypothyroidism. Furthermore, this decrease could be reversed by TRH, but not thyroid hormone replacement, suggesting a direct involvement of TRH in the regulation of thyrotrophs. The TRH−/− mice also exhibited hyperglycemia, which was accompanied by impaired insulin secretion in response to glucose. These findings indicate that TRH−/− mice provide a model of exploiting tertiary hypothyroidism, and that TRH gene abnormalities cause disturbance of insulin secretion resulting in marked hyperglycemia.

Complete sequencing of the Fugu WAGR region from WT1 to PAX6: Dramatic compaction and conservation of synteny with human chromosome 11p13

The pufferfish Fugu rubripes has a genome ≈7.5 times smaller than that of mammals but with a similar number of genes. Although conserved synteny has been demonstrated between pufferfish and mammals across some regions of the genome, there is some controversy as to what extent Fugu will be a useful model for the human genome, e.g., [Gilley, J., Armes, N. & Fried, M. (1997) Nature (London) 385, 305–306]. We report extensive conservation of synteny between a 1.5-Mb region of human chromosome 11 and <100 kb of the Fugu genome in three overlapping cosmids. Our findings support the idea that the majority of DNA in the region of human chromosome 11p13 is intergenic. Comparative analysis of three unrelated genes with quite different roles, WT1, RCN1, and PAX6, has revealed differences in their structural evolution. Whereas the human WT1 gene can generate 16 protein isoforms via a combination of alternative splicing, RNA editing, and alternative start site usage, our data predict that Fugu WT1 is capable of generating only two isoforms. This raises the question of the extent to which the evolution of WT1 isoforms is related to the evolution of the mammalian genitourinary system. In addition, this region of the Fugu genome shows a much greater overall compaction than usual but with significant noncoding homology observed at the PAX6 locus, implying that comparative genomics has identified regulatory elements associated with this gene.

Brain-derived neurotrophic factor-deficient mice develop aggressiveness and hyperphagia in conjunction with brain serotonergic abnormalities

Brain-derived neurotrophic factor (BDNF) has trophic effects on serotonergic (5-HT) neurons in the central nervous system. However, the role of endogenous BDNF in the development and function of these neurons has not been established in vivo because of the early postnatal lethality of BDNF null mice. In the present study, we use heterozygous BDNF+/− mice that have a normal life span and show that these animals develop enhanced intermale aggressiveness and hyperphagia accompanied by significant weight gain in early adulthood; these behavioral abnormalities are known to correlate with 5-HT dysfunction. Forebrain 5-HT levels and fiber density in BDNF+/− mice are normal at an early age but undergo premature age-associated decrements. However, young adult BDNF+/− mice show a blunted c-fos induction by the specific serotonin releaser-uptake inhibitor dexfenfluramine and alterations in the expression of several 5-HT receptors in the cortex, hippocampus, and hypothalamus. The heightened aggressiveness can be ameliorated by the selective serotonin reuptake inhibitor fluoxetine. Our results indicate that endogenous BDNF is critical for the normal development and function of central 5-HT neurons and for the elaboration of behaviors that depend on these nerve cells. Therefore, BDNF+/− mice may provide a useful model to study human psychiatric disorders attributed to dysfunction of serotonergic neurons.

A transgenic mouse model with an inducible skin blistering disease phenotype

One of the current limitations of gene transfer protocols involving mammalian genomes is the lack of spatial and temporal control over the desired gene manipulation. Starting from a human keratin gene showing a complex regulation as a template, we identified regulatory sequences that confer inducible gene expression in a subpopulation of keratinocytes in stratified epithelia of adult transgenic mice. We used this cassette to produce transgenic mice with an inducible skin blistering phenotype mimicking a form of epidermolytic hyperkeratosis, a keratin gene disorder. Upon induction by topical application of a phorbol ester, the mutant keratin transgene product accumulates in the differentiating layers of epidermis, leading to keratinocyte lysis after application of mechanical trauma. This mouse model will allow for a better understanding of the complex relationship between keratin mutation, keratinocyte cytoarchitecture, and hypersensitivity to trauma. The development of an inducible expression vector showing an exquisite cellular specificity has important implications for manipulating genes in a spatially and temporally controlled fashion in transgenic mice, and for the design of gene therapy strategies using skin as a tissue source for the controlled delivery of foreign substances.

Analysis of the Xenopus laevis CCAAT-enhancer binding protein α gene promoter demonstrates species-specific differences in the mechanisms for both auto-activation and regulation by Sp1

Transcription factors belonging to the CCAAT-enhancer binding protein (C/EBP) family have been implicated in the regulation of gene expression during differentiation, development and disease. Autoregulation is relatively common in the modulation of C/EBP gene expression and the murine and human C/EBPα genes have been shown to be auto-activated by different mechanisms. In the light of this finding, it is essential that autoregulation of C/EBPα genes from a wider range of different species be investigated in order to gauge the degree of commonality, or otherwise, that may exist. We report here studies that investigate the regulation of the Xenopus laevis C/EBPα gene (xC/EBPα). The –1131/+41 promoter region was capable of directing high levels of expression in both the human hepatoma Hep3B and the Xenopus kidney epithelial A6 cell lines, and was auto-activated by expression vectors specifying for xC/EBPα or xC/EBPβ. Deletion analysis showed that the –321/+41 sequence was sufficient for both the constitutive promoter activity and auto-activation and electrophoretic mobility shift assays identified the interaction of C/EBPs and Sp1 to this region. Although deletion of either the C/EBP or the Sp1 site drastically reduced the xC/EBPα promoter activity, multimers of only the C/EBP site could confer autoregulation to a heterologous SV40 promoter. These results indicate that, in contrast to the human promoter and in common with the murine gene, the xC/EBPα promoter was subject to direct autoregulation. In addition, we demonstrate a novel species-specific action of Sp1 in the regulation of C/EBPα expression, with the factor able to repress the murine promoter but activate the Xenopus gene.

A joint hazard and time scaling model to compare survival curves.

To provide a more general method for comparing survival experience, we propose a model that independently scales both hazard and time dimensions. To test the curve shape similarity of two time-dependent hazards, h1(t) and h2(t), we apply the proposed hazard relationship, h12(tKt)/ h1(t) = Kh, to h1. This relationship doubly scales h1 by the constant hazard and time scale factors, Kh and Kt, producing a transformed hazard, h12, with the same underlying curve shape as h1. We optimize the match of h12 to h2 by adjusting Kh and Kt. The corresponding survival relationship S12(tKt) = [S1(t)]KtKh transforms S1 into a new curve S12 of the same underlying shape that can be matched to the original S2. We apply this model to the curves for regional and local breast cancer contained in the National Cancer Institute's End Results Registry (1950-1973). Scaling the original regional curves, h1 and S1 with Kt = 1.769 and Kh = 0.263 produces transformed curves h12 and S12 that display congruence with the respective local curves, h2 and S2. This similarity of curve shapes suggests the application of the more complete curve shapes for regional disease as templates to predict the long-term survival pattern for local disease. By extension, this similarity raises the possibility of scaling early data for clinical trial curves according to templates of registry or previous trial curves, projecting long-term outcomes and reducing costs. The proposed model includes as special cases the widely used proportional hazards (Kt = 1) and accelerated life (KtKh = 1) models.

The kinesin walk: a dynamic model with elastically coupled heads.

Recently individual two-headed kinesin molecules have been studied in in vitro motility assays revealing a number of their peculiar transport properties. In this paper we propose a simple and robust model for the kinesin stepping process with elastically coupled Brownian heads that show all of these properties. The analytic and numerical treatment of our model results in a very good fit to the experimental data and practically has no free parameters. Changing the values of the parameters in the restricted range allowed by the related experimental estimates has almost no effect on the shape of the curves and results mainly in a variation of the zero load velocity that can be directly fitted to the measured data. In addition, the model is consistent with the measured pathway of the kinesin ATPase.

Glycoprotein 330/megalin: probable role in receptor-mediated transport of apolipoprotein J alone and in a complex with Alzheimer disease amyloid beta at the blood-brain and blood-cerebrospinal fluid barriers.

A soluble form of Alzheimer disease amyloid beta-protein (sA beta) is transported in the blood and cerebrospinal fluid mainly complexed with apolipoprotein J (apoJ). Using a well-characterized in situ perfused guinea pig brain model, we recently obtained preliminary evidence that apoJ facilitates transport of sA beta (1-40)-apoJ complexes across the blood-brain barrier and the blood-cerebrospinal fluid barrier, but the mechanisms remain poorly understood. In the present study, we examined the transport process in greater detail and investigated the possible role of glycoprotein 330 (gp330)/megalin, a receptor for multiple ligands, including apoJ. High-affinity transport systems with a Km of 0.2 and 0.5 nM were demonstrated for apoJ at the blood-brain barrier and the choroid epithelium in vivo, suggesting a specific receptor-mediated mechanism. The sA beta (1-40)-apoJ complex shared the same transport mechanism and exhibited 2.4- to 10.2-fold higher affinity than apoJ itself. Binding to microvessels, transport into brain parenchyma, and choroidal uptake of both apoJ and sA beta (1-40)-apoJ complexes were markedly inhibited (74-99%) in the presence of a monoclonal antibody to gp330/megalin and were virtually abolished by perfusion with the receptor-associated protein, which blocks binding of all known ligands to gp330. Western blot analysis of cerebral microvessels with the monoclonal antibody to gp330 revealed a protein with a mass identical to that in extracts of kidney membranes enriched with gp330/megalin, but in much lower concentration. The findings suggest that gp330/megalin mediates cellular uptake and transport of apoJ and sA beta (1-40)-apoJ complex at the cerebral vascular endothelium and choroid epithelium.

Active control of waves in a cochlear model with subpartitions.

Multiscale asymptotic methods developed previously to study macromechanical wave propagation in cochlear models are generalized here to include active control of a cochlear partition having three subpartitions, the basilar membrane, the reticular lamina, and the tectorial membrane. Activation of outer hair cells by stereocilia displacement and/or by lateral wall stretching result in a frequency-dependent force acting between the reticular lamina and basilar membrane. Wavelength-dependent fluid loads are estimated by using the unsteady Stokes' equations, except in the narrow gap between the tectorial membrane and reticular lamina, where lubrication theory is appropriate. The local wavenumber and subpartition amplitude ratios are determined from the zeroth order equations of motion. A solvability relation for the first order equations of motion determines the subpartition amplitudes. The main findings are as follows: The reticular lamina and tectorial membrane move in unison with essentially no squeezing of the gap; an active force level consistent with measurements on isolated outer hair cells can provide a 35-dB amplification and sharpening of subpartition waveforms by delaying dissipation and allowing a greater structural resonance to occur before the wave is cut off; however, previously postulated activity mechanisms for single partition models cannot achieve sharp enough tuning in subpartitioned models.