First Evidence of Axial Shape Asymmetry and Configuration Coexistence in

The excited states of N=44 ^{74}Zn were investigated via γ-ray spectroscopy following ^{74}Cu ß decay. By exploiting γ-γ angular correlation analysis, the 2_{2}^{+}, 3_{1}^{+}, 0_{2}^{+}, and 2_{3}^{+} states in ^{74}Zn were firmly established. The γ-ray branching and E2/M1 mixing ratios for transitions deexciting the 2_{2}^{+}, 3_{1}^{+}, and 2_{3}^{+} states were measured, allowing for the extraction of relative B(E2) values. In particular, the 2_{3}^{+}→0_{2}^{+} and 2_{3}^{+}→4_{1}^{+} transitions were observed for the first time. The results show excellent agreement with new microscopic large-scale shell-model calculations, and are discussed in terms of underlying shapes, as well as the role of neutron excitations across the N=40 gap. Enhanced axial shape asymmetry (triaxiality) is suggested to characterize ^{74}Zn in its ground state. Furthermore, an excited K=0 band with a significantly larger softness in its shape is identified. A shore of the N=40 "island of inversion" appears to manifest above Z=26, previously thought as its northern limit in the chart of the nuclides.

Prevalence of ventilation tubes in children with a tracheostomy tube.

OBJECTIVES: To estimate the prevalence of operative ear disease in pediatric patients with tracheostomy tubes, as well as to identify risk factors predictive of operative otologic interventions in this patient cohort. METHODS: We hypothesize that the prevalence of operative middle ear disease in patients with a tracheostomy tube is greater than that of the population at large. To validate our anecdotal observations, we queried the CHCA hospital database (PHIS) regarding the association between tympanostomy tube placements in children with tracheostomies. To further investigate, a retrospective chart review was undertaken at our regional tertiary care children's hospital to determine the frequency at which tympanostomy tubes were placed in children who have a tracheostomy. Risk factors were analyzed, applying independent samples t-tests and Pearson's Chi-square test. Univariate and multivariate logistic regression models were constructed to estimate odds ratios (OR) and 95% confidence intervals (CI) for predictors of operative ear disease. Institutional review board (IRB) approval was obtained. RESULTS: Of a population of 181 patients with tracheostomies, 37 (or 20%, 95% CI 15-26%) have undergone placement of ventilation tubes in the past 3 years. No statistically significant difference was noted with regards to gender or race. The operative group had an average age of 23.0 months at the time of tracheostomy, compared to 52.5 months in the non-operative group (p=0.0022). In addition, home living situation, term birth, and craniofacial abnormalities were more frequently observed in the operative versus the non-operative group. Multivariate logistic regression models revealed the same factors as predictors of operative ear disease. CONCLUSION: The presence of a tracheostomy is associated with an increased risk of requiring ventilation tube placement over the population at large. Risk factors for operative middle ear disease among these children include age at time of the tracheostomy, craniofacial abnormalities, term birth, and home living situation.

The impact of recent advances in immunology and cancer therapy on nuclear medicine.

The explosive expansion of knowledge in immunology in recent decades has already affected the research and practice of nuclear medicine in several ways. New hematopoietic cells have been isolated and their functions discovered, including hematopoietic stem cells and dendritic cells (DCs). Many new humeral factors have been found that have potent effects on cells, including cytokines, growth factors, and specialized proteins. Radiolabeled compounds are needed to follow the pharmacodynamics of the humeral factors and to follow the migration of mobile cells in animals and humans. In this article, only DCs, cytokines, and growth factors used clinically are discussed. DCs are essential for defense against infectious diseases. Autologous DCs cultured for a week and pulsed with tumor antigens have already proved highly immunogenic compared with other methods for activating cytotoxic T cells, and preliminary studies suggest that DCs are more potent for tumor cell killing than monoclonal antibodies. DCs, unfortunately, also play an important role in causing certain human diseases. In allograft transplants, residual donor DCs are responsible for the cellular rejection; if they could be eliminated, rejection could be prevented. These cells are also detrimental in rheumatoid arthritis, other autoimmune diseases, asthma, and chronic obstructive pulmonary disease. Cytokines such as interleukin-2 and such growth factors as granulocyte-macrophage colony-stimulating factor and granulocyte colony-stimulating factor, administered to patients with malignancies to alleviate the leukopenia of chemotherapy agents, frequently alter the tissue distribution of radiopharmaceuticals; these alterations may be confused with disease.

The bZIP-like motif of hnRNP C directs the nuclear accumulation of pre-mRNA and lethality in yeast.

The hnRNP C protein tetramer cooperatively binds 230 nt increments of pre-mRNA in vitro in a salt-resistant manner and is located along the length of vertebrate transcripts in vivo. Based on these and other findings it has been suggested that hnRNP C functions as a chaperonin to maintain long lengths of RNA topologically single-stranded and accessible to splicing factors. We report here that human C protein is lethal when expressed in the yeast Saccharomyces cerevisiae. Through a series of fluorescent immunolocalization studies, lethality was observed to be associated with the rapid nuclear accumulation of both C protein and yeast pre-mRNA. Studies using various protein constructs and the two hybrid assay reveal that these events are dependent on the basic 40 residue high-affinity RNA binding domain and its contiguous leucine zipper-like motif (the bZLM, residues 140-214). Additionally, equilibrium binding studies have shown that the bZLM is the determinant of C protein's salt-resistant RNA binding mode. Taken together, these findings further distinguish the bZIP-like domain as the major determinant of C protein's high-affinity interaction with RNA, oligomerization, and its highly cooperative RNA binding activity. Finally, these findings indicate that yeast and vertebrates may possess a conserved mechanism for general import of RNP although a true homolog to vertebrate C protein appears not to exist in yeast. Lethality is likely due to the absence in yeast of specific mechanisms for the removal of human C protein from nascent transcripts.

Effects of insulinlike growth factor binding proteins on insulinlike growth factor-I biodistribution in tumor-bearing nude mice.

UNLABELLED: This study evaluated the biodistribution and tumor targeting ability of radiolabeled insulinlike growth factor (IGF)-I. Because IGF binding proteins (IGFBPs) play a critical role in modulating IGF activity, the binding properties of 125I-labeled IGF-I to IGFBPs were investigated in vitro and in vivo. Because a large amount of the IGF-I was catabolized in vivo, we also studied the catabolism of IGF-I by tumor cells in vitro. METHODS: 125I-labeled-IGF-I was prepared using the chloramine T method. The biodistribution of 125I-labeled-IGF-I in tumor-bearing nude mice was compared between groups injected with 125I-labeled IGF-I alone or coinjected with unlabeled peptide. In vitro and in vivo chromatography studies were performed to evaluate the binding profile to IGFBPs and the degree of catabolites in serum as well as urine. RESULTS: Data indicated that the binding of radiolabeled IGF-I to IGFBPs in vitro was dose dependent. However, there was a difference in complex formation between the serum and the heparinized plasma. In heparinized plasma, the radioactivity shifted from a 30- to 50-kDa complex to a 150-kDa complex and to a free ligand, because the binding of heparin with IGFBPs decreased its affinity for IGF-I. In plasma prepared with acid citrate dextrose a binding pattern identical to that of serum was observed. Moreover, there was a binding difference between mouse and rat. The 125I-labeled IGF-I catabolized very quickly when incubated at 37 degrees C but not at all at 4 degrees C. In tumor-bearing nude mice, the uptake of radioactivity in normal tissues decreased quickly, particularly in the kidneys. In mice coinjected with unlabeled carrier, the radioactivity in most normal tissues was lower and the tumor uptake higher than in the mice without carrier. CONCLUSION: These data confirm that 125I-labeled IGF-I is avidly bound to IGFBPs, both in vitro and in vivo. By partially saturating this binding with unlabeled peptides, a favorable biodistribution was achieved, including faster clearance from normal tissue and higher tumor uptake, which resulted in better tumor-to-nontumor ratios. Nevertheless, the rapid catabolism and release of the radiolabel from tumor tissue result in a suboptimal targeting agent.

Evaluation practices for college students with LD.

To examine current practices in the use of psychoeducational evaluations for service delivery, we surveyed 91 service providers to college students with learning disabilities. The three purposes of the survey were to determine (a) whether service delivery decisions are based on information from psychoeducational evaluations, (b) which sections of the psychoeducational report are most useful in making service delivery decisions, and (c) the respondents' satisfaction with the tests and measurements for service delivery. The findings supported the common belief that data from psychoeducational evaluations serve as the primary basis for both eligibility and specific accommodation determinations. Respondents reported that all sections of the psychoeducational evaluation written report were useful, with the least useful section being test scores and the most useful being the summary of cognitive strengths and weaknesses. However, the section used most often for service delivery decisions was the professional's recommendations.

Oligonucleotide binding specificities of the hnRNP C protein tetramer.

Through the use of various non-equilibrium RNA binding techniques, the C protein tetramer of mammalian40S hnRNP particles has been characterized previously as a poly(U) binding protein with specificity for the pyrimidine-rich sequences that often precede 3' intron-exon junctions. C protein has also been characterized as a sequence-independent RNA chaperonin that is distributed along nascent transcripts through cooperative binding and as a protein ruler that defines the length of RNA packaged in 40S monoparticles. In this study fluorescence spectroscopy was used to monitor C protein-oligonucleotide binding in a competition binding assay under equilibrium conditions. Twenty nucleotide substrates corresponding to polypyrimidine tracts from IVS1 of the adenovirus-2 major late transcript, the adenovirus-2 oncoprotein E1A 3' splice site, IVS2 of human alpha-tropomyosin, the consensus polypyrimidine tract for U2AF65, AUUUA repeats and r(U)20were used as competitors. A 20 nt beta-globin intronic sequence and a randomly generated oligo were used as competitor controls. These studies reveal that native C protein possesses no enhanced affinity for uridine-rich oligonucleotides, but they confirm the enhanced affinity of C protein for an oligonucleotide identified as a high affinity substrate through selection and amplification. Evidence that the affinity of C protein for the winner sequence is due primarily to its unique structure or to a unique context is seen in its retained substrate affinity when contiguous uridines are replaced with contiguous guanosines.

Synthesis, in vitro binding, and tissue distribution of radioiodinated 2-[125I]N-(N-benzylpiperidin-4-yl)-2-iodo benzamide, 2-[125I]BP: a potential sigma receptor marker for human prostate tumors.

The preclinical evaluation of a sigma receptor-specific radiopharmaceutical that binds to human prostate tumor cells with a high affinity is described. We have synthesized and radioiodinated 2-[125I]-N-(N-benzylpiperidin-4-yl)-2-iodobenzamide (2-[125I]BP) that possesses high affinity for both sigma-1 and sigma-2 receptor subtypes that are expressed on a variety of tumor cells. 2-IBP was synthesized, purified and characterized by routine spectroscopic and analytical methods. Radioiodination was accomplished using an oxidative iododestannylation reaction in the presence of chloramine T in high yields (76%-93%) with a very high-specific activity (1700-1900 Ci/mmol). The in vitro competition binding studies of 2-[125I]BP with various sigma receptor ligands in LnCAP human prostate tumor cells showed a dose-dependent saturable binding. The inhibition constants (Ki, nM) for binding of 2-[125I]BP to human prostate tumor cells for 4-IBP, haloperidol and 2-IBP were 4.09, 6.34 and 1.6 nM, respectively. The clearance of 2-[125I]BP, in Sprague-Dawley rats, was rapid from the blood pool, other normal tissues and the total body. Tissue distribution studies in nude mice bearing human prostate tumor (DU-145) also showed a fast clearance from normal organs. The tumor had the highest percentage of injected dose per gram (%ID/g) of all tissues at 4 h as well as 24 h (2.0 +/- 0.05 and 0.147 +/- 0.038 ID/g, respectively) postinjection. The in vivo receptor binding specificity was demonstrated using haloperidol (a known high-affinity sigma receptor ligand). A significant decrease (> 50%, p = 0.001) was observed in tumor concentration when haloperidol was used as a blocking agent. The high affinity of 2-[125I]BP for sgma receptor-binding sites, its fast in vivo clearance from normal organs and its high uptake and retention in tumor implies that 2-[123I]BP or 2-[131I]BP may be a promising tracer for noninvasive imaging of human prostate tumors.

Multiple RRMs contribute to RNA binding specificity and affinity for polypyrimidine tract binding protein.

Polypyrimidine tract binding protein (PTB) is a 57 kD hnRNP protein (hnRNP I) that binds to the pyrimidine tract typically found near the 3' end of introns. Primary sequence analysis suggests that PTB contains four RNA recognition motifs (RRMs). Data from comparative structural and deletional analysis of PTB are consistent with the presence of a four reiterated domain structure. Since PTB exists in solution as a homodimer, it contains an oligomeric array of eight RRMs. Though the function of RRMs in a monomeric context has been addressed, the significance of their presence in an oligomeric context has not been investigated. To correlate structural motifs with function, we have analyzed the RNA binding properties of wild-type and deletion constructs of PTB that contain RRMs in both an oligomeric and monomeric context. These studies indicate that there is not a strong correlation between the RNA binding affinity and specificity upon oligomerization. However, the mode of RNA interaction and dimerization is linked. We have also found that the primary contributor to the free energy of PTB binding and the primary determinant for RNA binding specificity resides in RRM 3, while the primary contributor to dimer stabilization coincides with residues in RRM 2.

Biodistribution of 125I-labeled des(1-3) insulin-like growth factor I in tumor-bearing nude mice and its in vitro catabolism.

Insulin-like growth factor I (IGF-I) is a potent mitogen for many tumor cell lines, and IGF-I receptors are overexpressed in many tumors. Specific IGF-binding proteins (IGFBPs) modulate the interaction of IGF and its receptors. Consequently, radiolabeled IGF-I has been considered for tumor imaging. In the present study, we investigated the biodistribution of 125I-labeled des(1-3)IGF-I, a truncated analogue of IGF-I, in tumor-bearing nude mice. Additional studies included its catabolism by tumor cells in vitro and its binding to serum IGFBPs in vivo in nude mice. We also compared groups that were and were not injected with unlabeled peptide analogue. Our data showed that 125I-labeled des(1-3)IGF-I catabolized very fast, with a rapid appearance of nonprecipitable iodine, when incubated at 37 degrees C, but it was not catabolized at 4 degrees C incubation. 125I-labeled des(1-3)IGF-I was bound to serum-binding proteins, mainly in a complex with a molecular weight of M(r) 150,000. The uptake of radioactivity in normal tissues decreased quickly with time, particularly in the kidneys. In mice receiving higher doses of des(1-3)IGF-I, the radioactivity in all normal tissues was lower than in the mice with no carrier-added des(1-3)IGF-I, except in the stomach and spleen. These data suggest that 125I-labeled des(1-3)IGF-I is rapidly internalized after binding to the IGF receptor and is rapidly catabolized with release of breakdown products. Lower specific activity of 125I-labeled des(1-3)IGF-I resulted in altered biodistribution, including faster blood clearance and higher tumor uptake, by decreasing the formation of complexes with IGFBPs.

Mutation of PTB binding sites causes misregulation of alternative 3' splice site selection in vivo.

Alternative splicing of pre-mRNA is a commonly used mechanism to regulate gene expression in higher eukaryotes. However, with the exception of regulated cascades in Drosophila, the cis-acting elements and the trans-acting factors that control tissue- and/or developmentally regulated splicing remain largely unidentified. Cis-acting elements that control smooth muscle-specific repression of exon 3 of alpha-tropomyosin (alpha-TM) have been identified recently and consist of two regions that flank this exon. Deletion of either element causes misregulated splicing of alpha-TM in transfected smooth muscle cells. In experiments designed to characterize essential sequences within each element and the factors that interact with these sequences, we have identified two overlapping sequences within the downstream regulatory element (DRE) that are identical to binding sites for polypyrimidine tract binding protein (PTB) that were identified using iterative selection techniques. Mutation of these sites caused aberrant splicing regulation in transfected smooth muscle cells. In addition, sequences identical to high-affinity PTB binding sites were also detected upstream of exon 3 and mutation of these sites also resulted in misregulation of splicing in vivo, suggesting that PTB binding to specific sequences flanking exon 3 is responsible, in part, for the repression of exon 3. Consistent with this hypothesis, UV crosslinking and equilibrium binding assays confirm that the same mutations that cause misregulated splicing also disrupt PTB binding to RNA.

A major determinant of hnRNP C protein binding to RNA is a novel bZIP-like RNA binding domain.

The C protein tetramer of hnRNP complexes binds approximately 150-230 nt of RNA with high cooperativity (McAfee J et al., 1996, Biochemistry 35:1212-1222). Three contiguously bound tetramers fold 700-nt lengths of RNA into a 19S triangular intermediate that nucleates 40S hnRNP assembly in vitro (Huang M et al., 1994, Mol Cell Biol 14:518-533). Although it has been assumed that the consensus RNA recognition motif (RRM) of C protein (residues 8-87) is the primary determinant of RNA binding, we report here that a recombinant construct containing residues 1-115 has very low affinity for RNA at physiological ionic strength (100 mM NaCl). Moreover, we demonstrate that an N-terminal deletion construct lacking the consensus RRM but containing residues 140-290 binds RNA with an affinity sufficient to account for the total free energy change observed for the binding of intact protein. Like native C protein, the 140-290 construct is a tetramer in solution and binds RNA stoichiometrically in a salt-resistant manner in 100-300 mM NaCl. Residues 140-179 of the N-terminal truncated variant contain 11 basic and 2 acidic residues, whereas residues 180-207 specify a leucine zipper motif that directs dimer assembly. Elements within the 50-residue carboxy terminus of C protein are required for tetramer assembly. A basic region followed by a leucine zipper is identical to the domain organization of the basic-leucine zipper (bZIP) class of DNA binding proteins. Sequence homologies with other proteins containing RRMs and the bZIP motif suggest that residues 140-207 represent a conserved bZIP-like RNA binding motif (designated bZLM). The steric orientation of four high-affinity RNA binding sites about rigid leucine zipper domains may explain in part C protein's asymmetry, its large occluded site size, and its RNA folding activity.

Radiolabeled peptides and other ligands for receptors overexpressed in tumor cells for imaging neoplasms.

For better detection of neoplasms by scintigraphy, ligands that bind specifically to surface receptors overexpressed in tumor cells are being developed. These ligands must be labeled with the readily available radionuclides of iodine, [111In] or [99mTc], without blocking their interaction with the specific cell membrane receptors. The promising ligands include bioactive endogenous peptides or their analogues, inhibitors of glucose transport proteins, estrogen and sigma receptor ligands, growth factors, and cytokines.

Radioiodinated (aminostyryl)pyridinium (ASP) dyes: new cell membrane probes for labeling mixed leukocytes and lymphocytes for diagnostic imaging.

We prepared [125I/131I]iodo-(aminostyryl)pyridinium dyes from tributylstannyl precursors. ASP 7a and 7b labeled leukocytes ex vivo (70-94%) using saline with or without washing plasma from cells. Viability of peripheral blood lymphocytes (PBLs) (dogs, rats) and splenic lymphocytes (rats) labeled with 7a and 7b (71-82%) was unchanged after labeling (> or = 88%). Canine 7b-leukocytes showed higher uptake in inflammatory lesions than did 111In-oxine leukocytes. At 3 h, aspirates contained more radioiodine than 111In (1.65:1 to 22:1) and radioiodine was cell bound. ROI measurements (3 h) gave abscess to contralateral knee ratios of 12.3 and 10.6 for 131I-7b vs. 4.8 and 2.3 for 111In-oxine.

Equilibrium DNA binding of Sac7d protein from the hyperthermophile Sulfolobus acidocaldarius: fluorescence and circular dichroism studies.

The thermodynamics of the binding of the Sac7d protein of Sulfolobus acidocaldarius to double-stranded DNA has been characterized using spectroscopic signals arising from both the protein and the DNA. Ligand binding density function analysis has been used to demonstrate that the fractional change in protein intrinsic tryptophan fluorescence quenching that occurs upon DNA binding is equal to the fraction of protein bound. Reverse titration data have been fit directly to the McGhee-von Hippel model [McGhee, J., & von Hippel, P. (1974) J. Mol. Biol. 86, 469-489] using nonlinear regression. Sac7d binds noncooperatively to poly(dGdC) x poly(dGdC) with an intrinsic affinity of 6.5 x 10(6) M(-1) and a site size of 4 base pairs in 1 mM KH2PO4 and 50 mM KC1 (pH 6.8). Some binding sequence preference is noted, with the binding to poly(dIdC) x poly(dIdC) over 10-fold stronger than to poly(DAdT) x poly(dAdT). The binding is largely driven by the polyelectrolyte effect and is consistent with a release of 4.4 monovalent cations from DNA upon complex formation or the formation of 5 ion pairs at the protein-DNA interface. Extrapolation of salt back-titration data to 1 M KC1 indicates a -2.2 kcal/mol nonelectrostatic contribution to the binding free energy. A van't Hoff analysis of poly(dGdC) x poly(dGdC) binding shows that the binding enthalpy is approximately zero and the process is entropically driven. The affinity decreases slightly between pH 5.4 and 8.0. There is no significant difference between the binding parameters of recombinant Sac7d and native Sac7 proteins, indicating that methylation of the native protein has no effect on the DNA binding function. The binding of Sac7d to various DNAs leads to a significant increase in the DNA long-wavelength circular dichroism (CD) band, the intensity of which shows a sigmoidal dependence on Sac7d concentration. The sigmoidal CD binding isotherm can be quantitatively modeled by a conformational transition in the DNA that is cooperatively induced when protein monomers are bound within a given number of base pairs, ranging from zero for poly(dIdC) x poly(dIdC) to 8 or less for poly(dAdG) x poly(dCdT).

Proteins C1 and C2 of heterogeneous nuclear ribonucleoprotein complexes bind RNA in a highly cooperative fashion: support for their contiguous deposition on pre-mRNA during transcription.

Proteins C1 and C2 together comprise about one-third the protein mass of mammalian core 40S heterogeneous nuclear ribonucleoprotein particles (40S hnRNP) and exist as heterotetramers of (C1)3C2. On the basis of nonequilibrium binding studies, it has been suggested that the C proteins specifically bind oligo(U)- and poly(U)-rich sequences, and preferentially associate with uridine-rich regions near the 3' termini of many introns. We describe here a more quantitative characterization of the equilibrium binding properties of native and recombinant C protein to homoribopolymers using fluorescence spectroscopy. Like C protein from HeLa cells, the recombinant proteins spontaneously oligomerize to form tetramers with the same hydrodynamic properties as native protein. Near-stoichiometric binding titrations of the fluorescent homoribopolymer polyethenoadenosine (poly[r(epsilon A)]) with recombinant (C1)4 and (C2)4 homotetramers along with competition binding assays with poly(A) and poly(C) indicate that the binding site size (n) is between 150 and 230 nucleotides. This site size range is in close agreement with that previously determined for native C protein through hydrodynamic and ultrastructural studies (approximately 230 nucleotides). (C1)4 and (C2)4 bind poly(G) with intrinsic affinities (Ki) of 10(9) M-1, which are a hundredfold higher than their affinities for poly(U). In opposition to reports that C protein does not bind poly(A) and poly(C), we find that the C proteins bind these substrates with moderate Ki, but with high cooperativity (omega). The overall affinity (K omega) for the binding of both proteins to poly(A) and poly(C) is 10-fold higher (> 10(8) but < 10(9) M-1) than their affinities for poly(U). The highly cooperative binding of C protein to these substrates provides a mechanistic basis for the distribution of C protein along the length of nucleic acid substrates.