Generation and characterization of antibodies specific for caspase-cleaved neo-epitopes: a novel approach.

Apoptosis research has been significantly aided by the generation of antibodies against caspase-cleaved peptide neo-epitopes. However, most of these antibodies recognize the N-terminal fragment and are specific for the protein in question. The aim of this project was to create antibodies, which could identify caspase-cleaved proteins without a priori knowledge of the cleavage sites or even the proteins themselves. We hypothesized that many caspase-cleavage products might have a common antigenic shape, given that they must all fit into the same active site of caspases. Rabbits were immunized with the eight most prevalent exposed C-terminal tetrapeptide sequences following caspase cleavage. After purification of the antibodies we demonstrated (1) their specificity for exposed C-terminal (but not internal) peptides, (2) their ability to detect known caspase-cleaved proteins from apoptotic cell lysates or supernatants from apoptotic cell culture and (3) their ability to detect a caspase-cleaved protein whose tetrapeptide sequence differs from the eight tetrapeptides used to generate the antibodies. These antibodies have the potential to identify novel neo-epitopes produced by caspase cleavage and so can be used to identify pathway-specific caspase cleavage events in a specific cell type. Additionally this methodology may be applied to generate antibodies against products of other proteases, which have a well-defined and non-promiscuous cleavage activity.

Ex vivo purging by adenoviral p53 gene therapy does not affect NOD-SCID repopulating activity of human CD34+ cells.

Co-incubation of a replication-deficient, recombinant adenovirus carrying the wild-type p53 gene (rAd-p53) and hematopoietic stem cell (HSC) products from patients with breast cancer can significantly reduce tumor cell contamination. Whereas this approach provides a powerful tumor cell purging strategy, potential detrimental effects on the HSC population have not been investigated. The ability of human HSC to reconstitute hematopoiesis in severe combined immunodeficient (SCID) mice and to undergo secondary transplantation provides the only nonclinical measure of self-renewing, stem cell function. The objective of this study was to investigate whether co-incubation with rAd-p53 compromised the SCID repopulating activity (SRA) of HSC. Granulocyte colony-stimulating factor-mobilized human CD34+ cells were co-cultured with rAd-p53 at our targeted clinical dose, and the ability of these cells to establish multilineage hematopoiesis in sublethally irradiated, nonobese diabetic (NOD)-SCID mice was investigated. The persistence of human cells in the mice was investigated by flow cytometry, granulocyte-macrophage colony-forming unit assay, and polymerase chain reaction of human Alu sequences. Further, limiting dilution analysis provided a quantitative comparison between the SRA of CD34+ cells co-incubated with rAd-p53 and control CD34+ cells (no rAd-p53 co-incubation). We conclude that co-incubation with rAd-p53 has little effect on the SRA of HSC.

On the relationships between the fixed-f1, fixed-f2, and fixed-ratio phase derivatives of the 2f1-f2 distortion product otoacoustic emission.

For primary frequency ratios, f2/f1, in the range 1.1-1.3, the fixed-f1 ("f2-sweep") phase derivative of the 2f1-f2 distortion product otoacoustic emission (DPOAE) is larger than the fixed-f2("f1-sweep") one. It has been proposed by some researchers that part or all of the difference between these delays may be attributed to the so-called cochlear filter "build-up" or response time in the DPOAE generation region around the f2 tonotopic site. The analysis of an approximate theoretical expression for the DPOAE signal [Talmadge et al., J. Acoust. Soc. Am. 104, 1517-1543 (1998)] shows that the contributions to the phase derivatives associated with the cochlear filter response is small. It is also shown that the difference between the phase derivatives can be qualitatively accounted for by assuming the approximate scale invariance of cochlear mechanics. The effects of DPOAE fine structure on the phase derivative are also explored, and it is found that the interpretation of the phase derivative in terms of the phase variation of a single DPOAE component can be quite problematic.

The gene for a novel transmembrane protein containing epidermal growth factor and follistatin domains is frequently hypermethylated in human tumor cells.

A DNA fragment frequently hypermethylated in tumor cells was isolated using a novel screening strategy termed methylation-sensitive arbitrarily primed PCR. The isolated sequence corresponded to a CpG island at the 5' end of a previously unknown gene, TPEF (transmembrane protein containing epidermal growth factor and follistatin domains). Expression of TPEF was observed using Northern master blot analysis of a variety of normal tissues including colon, bladder, and prostate tissue. TPEF maps to human chromosome 2q33, where frequent loss of heterozygosity is seen in various human tumors, and TPEF was not expressed in most human colon and various other tumor cell lines examined by reverse transcription-PCR. Nine of 11 tumor cell lines were highly methylated in the 5' region and the first exon of the gene that demonstrated features characteristic of a CpG island. However the other two cell lines, which expressed TPEF, were hypomethylated in the 5' end of the gene. The region was also hypermethylated in 11 of 16 primary bladder tumors and in 3 of 4 primary colon tumors when compared with adjacent normal tissue. Our results suggest that potential tumor suppressor genes can be isolated from human tumors by virtue of their altered methylation patterns.

Modeling the temporal behavior of distortion product otoacoustic emissions.

The temporal behavior of the 2f1-f2 distortion product otoacoustic emission is theoretically investigated for the case in which the lower frequency (f1) primary tone is on continuously, and the higher frequency (f2) one is pulsed on and off [e.g., Talmadge et al., J. Acoust. Soc. Am. 105, 275-292 (1999)]. On physical grounds, this behavior is expected to be characterized by various group delays associated with the propagation of (1) the f2 cochlear primary wave between the cochlear base and the primary distortion product generation region around x2 (the f2 tonotopic place), and (2) the 2f1-f2 cochlear distortion product (DP) waves between the cochlear base, the primary generation region of the distortion product, and the region around the 2f1-f2 tonotopic place where the generated apical moving DP wave is reflected toward the cochlear base [e.g., Talmadge et al., J. Acoust. Soc. Am. 104, 1517-1543 (1998)]. An approximate analytic expression is obtained for this behavior from the analysis of the Fourier integral representation of the auditory peripheral response to the primary stimuli. This expression also approximately describes the transient build-up of the components of different latencies in terms of the damping properties of the cochlear partition. It is shown that considerable caution must be applied in attempting to relate phase derivatives of the distortion product otoacoustic emissions for steady state stimuli and the physical time delays which are associated with the temporal behavior of a distortion product emission in the case of a pulsed primary.

Interrelations among distortion-product phase-gradient delays: their connection to scaling symmetry and its breaking.

Distortion-product-otoacoustic-emission (DPOAE) phase-versus-frequency functions and corresponding phase-gradient delays have received considerable attention because of their potential for providing information about mechanisms of emission generation, cochlear wave latencies, and characteristics of cochlear tuning. The three measurement paradigms in common use (fixed-f1, fixed-f2, and fixed-f2/f1) yield significantly different delays, suggesting that they depend on qualitatively different aspects of cochlear mechanics. In this paper, theory and experiment are combined to demonstrate that simple phenomenological arguments, which make no detailed mechanistic assumptions concerning the underlying cochlear mechanics, predict relationships among the delays that are in good quantitative agreement with experimental data obtained in guinea pigs. To understand deviations between the simple theory and experiment, a general equation is found that relates the three delays for any deterministic model of DPOAE generation. Both model-independent and exact, the general relation provides a powerful consistency check on the measurements and a useful tool for organizing and understanding the structure in DPOAE phase data (e.g., for interpreting the relative magnitudes and intensity-dependencies of the three delays). Analysis of the general relation demonstrates that the success of the simple, phenomenological approach can be understood as a consequence of the mechanisms of emission generation and the approximate local scaling symmetry of cochlear mechanics. The general relation is used to quantify deviations from scaling manifest in the measured phase-gradient delays; the results indicate that deviations from scaling are typically small and that both linear and nonlinear mechanisms contribute significantly to these deviations. Intensity-dependent mechanisms contributing to deviations from scaling include cochlear-reflection and wave-interference effects associated with the mixing of distortion- and reflection-source emissions (as in DPOAE fine structure). Finally, the ratio of the fixed-f1 and fixed-f2 phase-gradient delays is shown to follow from the choice of experimental paradigm and, in the scaling limit, contains no information about cochlear physiology whatsoever. These results cast considerable doubt on the theoretical basis of recent attempts to use relative DPOAE phase-gradient delays to estimate the bandwidths of peripheral auditory filters.

Modeling the combined effects of basilar membrane nonlinearity and roughness on stimulus frequency otoacoustic emission fine structure.

A theoretical framework for describing the effects of nonlinear reflection on otoacoustic emission fine structure is presented. The following models of cochlear reflection are analyzed: weak nonlinearity, distributed roughness, and a combination of weak nonlinearity and distributed roughness. In particular, these models are examined in the context of stimulus frequency otoacoustic emissions (SFOAEs). In agreement with previous studies, it is concluded that only linear cochlear reflection can explain the underlying properties of cochlear fine structures. However, it is shown that nonlinearity can unexpectedly, in some cases, significantly modify the level and phase behaviors of the otoacoustic emission fine structure, and actually enhance the pattern of fine structures observed. The implications of these results on the stimulus level dependence of SFOAE fine structure are also explored.

The human DNA methyltransferases (DNMTs) 1, 3a and 3b: coordinate mRNA expression in normal tissues and overexpression in tumors.

DNA methylation in mammals is required for embryonic development, X chromosome inactivation and imprinting. Previous studies have shown that methylation patterns become abnormal in malignant cells and may contribute to tumorigenesis by improper de novo methylation and silencing of the promoters for growth-regulatory genes. RNA and protein levels of the DNA methyltransferase DNMT1 have been shown to be elevated in tumors, however murine stem cells lacking Dnmt1 are still able to de novo methylate viral DNA. The recent cloning of a new family of DNA methyltransferases (Dnmt3a and Dnmt3b) in mouse which methylate hemimethylated and unmethylated templates with equal efficiencies make them candidates for the long sought de novo methyltransferases. We have investigated the expression of human DNMT1, 3a and 3b and found widespread, coordinate expression of all three transcripts in most normal tissues. Chromosomal mapping placed DNMT3a on chromosome 2p23 and DNMT3b on chromosome 20q11.2. Significant overexpression of DNMT3b was seen in tumors while DNMT1 and DNMT3a were only modestly over-expressed and with lower frequency. Lastly, several novel alternatively spliced forms of DNMT3b, which may have altered enzymatic activity, were found to be expressed in a tissue-specific manner.

Experimental confirmation of the two-source interference model for the fine structure of distortion product otoacoustic emissions.

High-resolution measurements of distortion product otoacoustic emissions (DPOAEs) from three different experimental paradigms are shown to be in agreement with the implications of a realistic "two-source" cochlear model of DPOAE fine structure. The measurements of DPOAE amplitude and phase imply an interference phenomenon involving one source in the region of strong nonlinear interaction of the primary waves (the strong "overlap" or generation region), and the other source region around the DPOAE tonotopic place. The component from the DPOAE place can be larger than the one from the generator region. These findings are supported by the analysis of the onset and offset of the DPOAE when the higher-frequency primary is pulsed on and off. The two-source hypothesis was further tested by adding a third tone closer in frequency to the DPOAE which modifies the amplitude of the component from the DPOAE place and leaves the one from the generator region unchanged. The results agree well with the model prediction that the variation with frequency, and implied latency, of the phase of the DPOAE tonotopic-place component are greater than the corresponding quantities for the component from the generation region.

Chromosomal mapping of three human LAMMER protein-kinase-encoding genes.

The eukaryotic LAMMER protein kinase family is encoded by at least three loci in the human genome, designated CLK1, 2, and 3. We have mapped these loci to 2q33, 1q21, and 15q24, respectively, by fluorescent in situ hybridization. Additionally, a CLK2 pseudo-gene has been located to 7p15-21.

Modeling otoacoustic emission and hearing threshold fine structures.

A class of cochlear models which account for much of the characteristic variation with frequency of human otoacoustic emissions and hearing threshold microstructure is presented. The models are based upon wave reflections via distributed spatial cochlear inhomogeneities and tall and broad cochlear activity patterns, as suggested by Zweig and Shera [J. Acoust. Soc. Am. 98, 2018-2047 (1995)]. They successfully describe in particular the following features: (1) the characteristic quasiperiodic frequency variations (fine structures) of the hearing threshold, synchronous and click-evoked emissions, distortion-product emissions, and spontaneous emissions; (2) the relationships between these fine structures; and (3) the distortion product emission filter shape. All of the characteristic frequency spacings are approximately the same (0.4 bark) and are mainly determined by the phase behavior of the apical reflection function. The frequency spacings for spontaneous emissions and threshold microstructure are predicted to be the same, but some deviations from these values are predicted for synchronous and click-evoked and distortion-product emissions. The analysis of models is aided considerably by the use of the solutions of apical, and basal, moving solutions (basis functions) of the cochlear wave equation in the absence of inhomogeneities.

Mutation of a gene encoding a protein with extracellular matrix motifs in Usher syndrome type IIa.

Usher syndrome type IIa (OMIM 276901), an autosomal recessive disorder characterized by moderate to severe sensorineural hearing loss and progressive retinitis pigmentosa, maps to the long arm of human chromosome 1q41 between markers AFM268ZD1 and AFM144XF2. Three biologically important mutations in Usher syndrome type IIa patients were identified in a gene (USH2A) isolated from this critical region. The USH2A gene encodes a protein with a predicted size of 171.5 kilodaltons that has laminin epidermal growth factor and fibronectin type III motifs; these motifs are most commonly observed in proteins comprising components of the basal lamina and extracellular matrixes and in cell adhesion molecules.

Ear canal reflectance in the presence of spontaneous otoacoustic emissions. I. Limit-cycle oscillator model.

Allen et al. [Abstract in Eighteenth Midwinter Research Meeting of the Association for Research in Otolaryngology, Des Moines, IA (1995)] have found that the ear canal reflectance passes through a minimum around the frequency of a spontaneous otoacoustic emission (SOAE). They considered this result to constitute evidence against active nonlinear cochlear function as the basis for SOAEs. In order to investigate theoretically the expected behavior of ear canal reflectance in the neighborhood of a SOAE associated with an active-nonlinear cochlea, we use a simplified model in which the ear drum end of the ear canal is effectively terminated by a nonlinear-active element. Under the influence of a sinusoidal driver at the entrance of the ear canal, this element will, to a good approximation, either (1) oscillate at both the frequency of the driver (at which the reflectance is determined) and the SOAE (at a suppressed level, corresponding to nonentrainment), or (2) be entrained and only oscillate at the driving frequency. The magnitude of the nonlinear ear canal reflectance is found to exceed unity only at sufficiently low stimulus levels, and occurs under conditions of entrainment and nonentrainment of the spontaneous emission. Otherwise, the reflectance is less than unity and, as a function of frequency, has a minimum around the SOAE frequency.

Spontaneous otoacoustic emission frequency is modulated by heartbeat.

Detailed analysis of spontaneous otoacoustic emissions (SOAEs) in human subjects revealed that all stable SOAEs sufficiently above the noise floor to permit appropriate analysis have sidebands at multiples of approximately 1 Hz. This is consistent with the hypothesis that SOAEs are modulated by heartbeat. Simultaneous measurement of the rate of blood flow to the thumb and the separation of the spectral sidebands demonstrated that they covary (r = 0.982, p < 5 x 10(-10)). An adaptive least-squares fit (LSF) paradigm was developed to facilitate the measurement of the instantaneous frequency and amplitude of the signals. A combination of traditional spectral analyses and new LSF analyses showed that the sideband generation stems from frequency modulation of the emissions. If there is any amplitude modulation correlated with the blood flow, it is below the noise floor of the analysis. The frequency of the emission was at a minimum when the blood flow was maximal. Examination of alternative mechanisms using computer simulations suggests that these changes stem from changes of 10-100 ppm in the mass of the basilar membrane.

Isolation of a novel human homologue of the gene coding for echinoderm microtubule-associated protein (EMAP) from the Usher syndrome type 1a locus at 14q32.

Usher syndrome type 1 (USH1) is an autosomal recessive, genetically heterogeneous disorder causing severe congenital deafness, retinitis pigmentosa, and vestibular dysfunction. The USHla locus located on 14q32 has been linked to the genetic markers D14S250 and D14S78. Using D14S250 and D14S78, we have isolated two nonchimeric YACs, 878g10 and 844g2, and a single BAC (135i20) and PAC (194e17) clone and have arranged them into a contig spanning over the D14S250 and D14S78 markers. The analysis of the YACs, BAC, and PAC revealed that the physical distance between D14S250 and D14S78 is less than 25 kb. Iterative cDNA library screening initiated with the EST 219670 found in the vicinity of the D14S78 marker yielded a cDNA contig. The nucleotide sequence of the cDNA encodes a protein of 717 amino acids in length, showing a high level of homology to the Echinoderm 77-kDa microtubule-associated protein (EMAP). The human homologue of Echinoderm microtubule-associated protein defines a novel human gene. We propose that the human EMAP is a strong candidate for the USH1a gene based on its genomic location and the proposed function of the protein.

The genomic structure of the gene defective in Usher syndrome type Ib (MYO7A).

Usher syndrome type Ib is a recessive autosomal disorder manifested by congenital deafness, vestibular dysfunction, and progressive retinal degeneration. Mutations in the human myosin VIIa gene (MYO7A) have been reported to cause Usher type Ib. Here we report the genomic organization of MYO7A. An STS content map was determined to discover the YAC clones that would cover the critical region for Usher syndrome type Ib. Three of the YACs (802A5, 966D6, and 965F10) were subcloned into cosmids and used to assemble a preliminary cosmid contig of the critical region. Part of the gene encoding human myosin VIIa was found in the preliminary cosmid contig. A cosmid, P1, PAC, and long PCR contig that contained the entire MYO7A gene was assembled. Primers were designed from the composite cDNA sequence and used to detect intron-exon junctions by directly sequencing cosmid, P1, PAC, and genomic PCR DNA. Alternatively spliced products were transcribed from the MYO7A gene: the largest transcript (7.4 kb) contains 49 exons. The MYO7A gene is relatively large, spanning approximately 120 kb of genomic DNA on chromosome 11q13.

A yeast artificial chromosome (YAC) contig encompassing the critical region of the X-linked lymphoproliferative disease (XLP) locus.

X-linked lymphoproliferative disease (XLP) is characterized by a marked vulnerability to Epstein-Barr virus (EBV) infection. Infection of XLP patients with EBV invariably results in fatal mononucleosis, agammaglobulinemia, or malignant lymphoma. Initially the XLP gene was assigned to a 10-cM region in Xq25 between DXS42 and DXS37. Subsequently, an interstitial, cytogenetically visible deletion in Xq25 was identified in one XLP family, 43. In this study we estimated the deletion in XLP patient 43-004 by dual-laser flow karyotyping to involve 2% of the X chromosome, or approximately 3 Mb of DNA sequence. From a human chromosome Xq25-specific yeast artificial chromosome (YAC) sublibrary, five YACs containing DNA sequences deleted in patient 43-004 have been isolated. Sequence-tagged sites (STSs) from these YACs have been used to identify interstitial deletions in unrelated XLP patients. Three more families with interstitial deletions were found. Two of the patients (63-003 and 73-032) carried an interstitial deletion of 3.0 Mb overlapping the 43-004 deletion. In one XLP patient (30-011) who exhibited the characteristic postinfectious mononucleosis phenotype of XLP with hypogammaglobulinemia and malignant lymphoma, a deletion of approximately 250 kb was detected overlapping the deletion detected in patients 43-004, 63-003, and 73-032. A YAC contig of 2.2 Mb spanning the XLP critical region, whose orientation on chromosome X was determined by double-color fluorescence in situ hybridization and which consists of 15 overlapping YAC clones, has been constructed. A detailed restriction enzyme map of the region has been constructed. YAC insert sizes were determined by counter-clamped homogenous electric field gel electrophoresis. Chimerism of YACs was determined by FISH and restriction mapping. On the basis of lambda subclones, YAC end-derived plasmids, and STSs with an average spacing of 100 kb, a long-range physical map was constructed using 5 rare-cutter restriction enzymes. The STSs and lambda subclones were used in Southern hybridization and PCR analyses. The work presented here substantially refines the critical region for XLP. The YAC contig with the overlapping interstitial deletions constitutes the basis for the construction of a transcriptional map of the critical region and facilitates the identification of the XLP gene.

Relaxation dynamics of spontaneous otoacoustic emissions perturbed by external tones. III. Response to a single tone at multiple suppression levels.

The relaxation dynamics of spontaneous otoacoustic emissions (SOAEs) interacting with an external tone have been successfully described using a van der Pol limit cycle oscillator model [Murphy et al., J. Acoust. Soc. Am. 97, 3702-3710 (1995) and Murphy et al., J. Acoust. Soc. Am. 97, 3711-3720 (1995)]. Data were collected for an SOAE interacting with a single-frequency ipsilateral suppressor. Transitions between different suppressed states were achieved by adding or removing signal at the suppressor frequency. The relaxation dynamics of the van der Pol model provided a good fit to the data.

The construction of a yeast artificial chromosome (YAC) contig in the vicinity of the Usher syndrome type IIa (USH2A) gene in 1q41.

The gene for Usher syndrome type II (USH2A), an autosomal recessive syndromic deafness, has been mapped to a region of 1q41 flanked proximally by D1S217 and distally by D1S439. Using sequence-tagged sites (STSs) within the region, a total of 21 yeast artificial chromosome (YAC) clones were isolated and ordered into a single contig that spans approximately 11.0 Mb. The order of microsatellite and STS markers in this region was established as D1S505-D1S425-DXS217-D1S556-D1S237-D1S4 74-EB1-EB2-KB6-AFM144XF2-KB1-K B4-D1S229-D1S490-D1S227-TGFbeta2-D1S439. Analysis of newly positioned polymorphic markers in recombinant individuals in two Usher syndrome type IIa families has enabled us to identify DXS474 and AFM144XF2 as two flanking markers for the Usher type IIa locus. The physical distance between the two markers is 1.0 Mb. This region is covered by eight YACs from the CEPH library: 945f7, 867g9, 762a6, 919h3, 794b8, 785h4, 848b9, and 841g2. A long-range physical map of the Usher type IIa critical region, using MluI, BssHII, NotI, EagI, and SacII, has been developed.