ErbB-4 ribozymes abolish neuregulin-induced mitogenesis

The epidermal growth factor-like receptor tyrosine kinase (ErbB) family is frequently overexpressed in a variety of human carcinomas, including breast cancer. To assist in characterizing the role of ErbB-4 in breast cancer, we generated three specific hammerhead ribozymes targeted to the ErbB-4 mRNA. These ribozymes, Rz6, Rz21, and Rz29, efficiently catalyzed the specific cleavage of ErbB-4 message in a cell-free system. We demonstrated that the neuregulin-induced mitogenic effect was abolished in ribozyme Rz29- and Rz6-transfected 32D/ErbB-4 cells. Inhibition of mitogenesis was characterized by ribozyme-mediated down-regulation of ErbB-4 expression. In addition, we provide the first evidence that different threshold levels of ErbB-4 expression and activation correlate with different responses to neuregulin stimulation. High levels of ErbB-4 expression, phosphorylation, and homodimerization are necessary for neuregulin-stimulated, interleukin 3-independent cell proliferation in the 32D/E4 cells. In the case of Rz29-transfected 32D/E4 cells, low levels of ErbB-4 expression allowed neuregulin-induced phosphorylation but were insufficient to couple the activated receptor to cellular signaling. Furthermore, expression of the functional ErbB-4 ribozyme in T47D human breast carcinoma cells led to a down-regulation of endogenous ErbB-4 expression and a reduction of anchorage-independent colony formation. These studies support the use of ErbB-4 ribozymes to define the role of ErbB-4 receptors in human cancers.     

Spectroscopic comparisons of the pH dependencies of Fe-substituted (Mn)superoxide dismutase and Fe-superoxide dismutase

We have compared the active sites of Escherichia coli Fe-substituted (Mn)superoxide dismutase [Fe-sub-(Mn)SOD] and Fe-SOD to elucidate the basis for the inactivity of Fe-sub-(Mn)SOD, despite its apparent similarity to Fe-SOD. The active site of (reduced) Fe2+-sub-(Mn)SOD is qualitatively similar to that of native Fe2+-SOD, indicating similar active site structures and coordination environments for Fe2+. Its nativelike pK is indicative of nativelike local electrostatics, and consistent with Fe2+-sub-(Mn)SOD's retention of ability to reduce O2*- [Vance and Miller (1998) J. Am. Chem. Soc. 120(3), 461-467]. The active site of (oxidized) Fe3+-sub-(Mn)SOD differs from that of Fe3+-SOD with respect to the EPR signals produced at both neutral and high pH, indicating different coordination environments for Fe3+. Although Fe3+-sub-(Mn)SOD binds the small anions N3- and F-, the KD for N3- is tighter than that of Fe3+-SOD, suggesting that the (Mn)SOD protein favors anion binding more than does the (Fe)SOD protein. The EPR spectral consequences of binding F- are reminiscent of those observed upon binding the first F- to Fe3+-SOD, but the EPR spectrum obtained upon binding N3- is different, consistent with crystallographic observation of a different binding mode for N3- in Thermus thermophilus Mn-SOD than Fe-SOD [Lah, M., et al. (1995) Biochemistry 34, 1646-1660]. We find a pK of 8.5 to be associated with dramatic changes in the EPR spectrum. In addition, we confirm the pK between 6 and 7 that has previously been reported based on changes in the optical signal and N3- binding [Yamakura, F., et al. (1995) Eur. J. Biochem. 227, 700-706]. However, this latter pK appears to be associated with much subtler changes in the EPR spectrum. The non-native pKs observed in Fe3+-sub-(Mn)SOD and the differences in the Fe3+ coordination indicated by the EPR spectra are consistent with Fe3+-sub-(Mn)SOD's inability to oxidize O2*- and suggest that its low E degrees is due to perturbation of the oxidized state.     

Comparison of various interpositional materials in the prevention of transphyseal bone bridge formation

Various interpositional materials, except muscle, have been used to prevent transphyseal bone bridge formation after resection of the damaged physeal plate. In this animal model, muscle was used as an interpositional material, and its effectiveness was compared with that of 3 known materials (fat, physeal allograft, and iliac apophyseal autograft). Five experiments were done on the distal femoral physis of 40 skeletally immature 3-month-old New Zealand white rabbits. The rabbits were divided into 5 groups, each containing 8 rabbits. A standard defect was created in the lateral distal physis of the left femur in all the rabbits. In Group A, there was no interpositional material. Vastus lateralis muscle, groin fat, physeal allograft, and iliac apophyseal autograft were inserted into the femoral defect in Groups B, C, D, and E, respectively. The right femur served as a sham control for the animals. The animals were sacrificed at 12 weeks after surgery. The results of limb length discrepancy and angular deformity of the groups with interpositional material were compared with those of Group A (experimental control). Muscle, fat, and iliac apophyseal autografts had less severe limb length discrepancy and angular deformity. These differences were statistically significant, whereas the differences between allograft and experimental control were statistically insignificant.     

Enhancement of radiation-induced hepatic microsomal epoxide hydrolase gene expression by oltipraz in rats

The effects of radiation exposure in conjunction with oltipraz, a chemopreventive agent, on the expression of the gene encoding hepatic microsomal epoxide hydrolase (mEH) were examined in rats. Rats exposed to a single dose of 3 Gy gamma rays exhibited timerelated changes in the hepatic mEH mRNA level. Whereas the mEH mRNA level was transiently decreased at 3 and 8 h after irradiation, the mRNA levels were increased 3- to 4-fold at 15 to 48 h postirradiation, returning to the level in untreated animals at 72 h. Treatment of rats with oltipraz resulted in 1- to 19-fold increases in hepatic mEH mRNA levels 24 h post-treatment at doses of 5-200 mg/kg. Although treatment with oltipraz at a dose of 30 mg/kg affected the mEH mRNA level minimally (i.e. approximately 2-fold), 3 Gy whole-body irradiation along with oltipraz treatment resulted in a 9-fold increase in the mEH mRNA level at 24 h post-treatment. Treatment of animals with both oltipraz and 3 Gy gamma radiation for 3 consecutive days resulted in a 7-fold increase in mEH mRNA, showing that the increases in mEH mRNA were enhanced by the combination treatment. In rats irradiated with 3 Gy for 5 consecutive days, however, the mEH mRNA level failed to increase due to cell injury. Studies were further designed to assess the effects of 0.5 Gy ionizing radiation and concomitant oltipraz treatment. RNA blot analysis showed that mEH mRNA levels failed to be significantly altered at 3, 8, 15, 24 and 48 h after a single dose of 0.5 Gy. Nonetheless, exposure of animals to 0.5 Gy daily for 3 to 5 consecutive days caused a 3-fold elevation in the hepatic mEH mRNA level. Furthermore, treatment of animals with both oltipraz (30 mg/kg/day) and 0.5 Gy of gamma rays resulted in an enhanced elevation in the mEH mRNA level at 24 h post-treatment compared to the individual treatment, resulting in a 7-fold relative increase. The enhanced expression of hepatic mEH mRNA by 0.5 Gy gamma radiation and oltipraz was also observed after treatment for 3 to 5 days (8- to 6-fold relative increases). Western immunoblot analyses showed that hepatic microsomes produced from the rats treated with 0.5 Gy daily for 3 to 5 days resulted in a approximately 2-fold induction of hepatic mEH and that rats exposed to radiation in combination with oltipraz showed 3-fold increases in the liver mEH protein. Thus the relative increase in mEH mRNA levels was consistent with the expression of the protein. These results demonstrate that ionizing radiation causes alterations in hepatic mEH gene expression with the induction of the protein and that the mEH gene expression is enhanced by oltipraz treatment.     

The occurrence of late asthmatic response to exercise after allergen challenge

OBJECTIVES: To evaluate the correlation between the pathological findings of stereotactic core needle biopsy (SCNB) and the prebiopsy mammographic findings, as well as the pathological findings of lesions that were subsequently removed by surgical excision. DESIGN: A retrospective review of 97 consecutive patients who underwent 100 SCNBs of suspicious nonpalpable mammographic lesions. The criterion standard is surgical excisional biopsy with needle localization. Mammographic findings were graded according to the American College of Radiology Breast Imaging Reporting and Data System. The pathological findings of SCNB were categorized into 4 groups: benign and specific, benign and nonspecific, premalignant, and malignant. Surgical excision of the lesion was performed if the pathological finding on SCNB was nonconcordant with the prebiopsy mammogram and when premalignant or malignant lesions were found. The pathological findings of lesions that were subsequently removed by surgical excision were compared with those of SCNB. SETTING: Community-based private multispecialty ambulatory practice. PATIENTS: A population-based sample composed of 97 patients who had grade III, IV, or V lesions on routine screening mammograms. INTERVENTION: Stereotactic core needle biopsy of nonpalpable mammographic lesions. MAIN OUTCOME MEASURES: Percentage of patients whose SCNB results were concordant with the mammographic findings and the pathological findings on subsequent surgical excision. RESULTS: Concordance between SCNB and mammography occurred in 97% of biopsy specimens. Concordance between the pathological findings of SCNB and those of surgically excised lesions occurred in 92.5% of biopsy specimens. We had 1 false-negative result. We had no false-positive diagnosis of cancer with SCNB. CONCLUSION: On the basis of accumulating literature and our own initial experience, SCNB is a promising, safe, and cost-effective procedure.     

Stabilized analogs of thymopentin. 1. 4,5-Ketomethylene pseudopeptides

The pentapeptide, thymopentin (Arg1-Lys2-Asp3-Val4-Tyr5) is known for its activity as an immunomodulating drug, but with limited half-life in plasma. In this first paper of a series of three studies, the synthesis of analogs stabilized at the peptide bond between the C-terminal amino acids via insertion of a ketomethylene moiety is described. N-Blocked pseudopeptides containing Val(k)Phe, Ala(k)Phe, and Val(k)Val units were prepared and attached to chloromethyl Merrifield resin via the carboxy terminal. Removal of the N-BOC group by trifluoroacetic acid was followed by sequential coupling with N-BOC dipeptides of aspartic acid to yield resin-bound N-BOC pseudotetrapeptides. Removal of N-BOC and coupling with N-BOC-r-N-tosylarginine followed by total cleavage of blocking groups and resin by HF afforded the target pseudopentapeptides. The analogs were found to compete favorably with thymopentin for binding to CEM cells, but binding was reduced by about 20-30% on average. All analogs showed significant enhancement of half-life versus thymopentin in mouse serum, but most showed only modest improvement in human serum. Insertion of proline or norleucine at position 2 in the chain caused a substantial increase in half-life (3-4-fold), while N-methylnorleucine conferred complete stability in the analogs.     

Subdoctoral Psychologists.

Comments on P. J. Woods' article on APA's concern with the Master's degree in psychology (American Psychologist, 1971, 26, 696-707). The present author suggests that before many more hours are expended in deciding whether subdoctoral psychologists should be called psychologists, the assumption that clinical practitioners holding a doctorate in psychology are better equipped than those trained at the master's level should be demonstrated. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Judging personality assessments: putting the Barnum report in perspective

The ability of expert and naive judges to discriminate between genuine and Barnum assessment statements was assessed. In a 2 X 2 X 2 design (naive vs. expert judges, genuine vs. Barnum test statements, sex), judges rated assessment statements for their information value, usefulness, social desirability, and typicalness. Results indicated that judges were able to make expected discriminations between genuine and Barnum statements. These results were discussed in terms of previous findings which have suggested that judges have seen Barnum statements as "accurate" or "good" as genuine statements. In the present study, the discriminations seemed due to the use of a population of judges more representative of clinical assessment consumers and to the more specific judgments required.     

Endothelial modulation of neural sympathetic vascular tone in canine skeletal muscle

The effect of nitric oxide synthase (NOS) inhibition and endothelin-A (ETA)-receptor blockade on neural sympathetic control of vascular tone in the gastrocnemius muscle was examined in anesthetized dogs under conditions of constant flow. Muscle perfusion pressure (MPP) was measured before and after NOS inhibition (Nomega-nitro-L-arginine methyl ester; L-NAME) and ETA-receptor blockade [cyclo-(D-Trp-d-Asp-Pro-D-Val-Leu); BQ-123]. Zero and maximum sympathetic nerve activities were achieved by sciatic nerve cold block and stimulation, respectively. In group 1 (n = 6), MPP was measured 1) before nerve cold block, 2) during nerve cold block, and 3) during nerve stimulation. Measurements under these conditions were repeated after L-NAME and then BQ-123. The same protocol was followed in group 2 (n = 6) except that the order of L-NAME and BQ-123 was reversed. MPP and muscle vascular resistance (MVR) increased after L-NAME and then decreased to control values after BQ-123. MVR decreased after BQ-123 alone and, with the addition of L-NAME, increased to a level not different from that observed during the control period. MVR fell during nerve cold block. This response was not affected by administration of L-NAME followed by BQ-123, but it was attenuated by administration of BQ-123 before L-NAME. The constrictor response during sympathetic nerve stimulation was enhanced by L-NAME; no further effect was observed with BQ-123, nor was the response affected when BQ-123 was given first. These findings indicate that endothelin contributes to 1) basal vascular tone in skeletal muscle and 2) the increase in skeletal muscle vascular resistance after NOS inhibition. Finally, nitric oxide "buffers" the degree of constriction in skeletal muscle vasculature during maximal sympathetic stimulation.