Expression of constitutively active Raf-1 in the mitochondria restores antiapoptotic and leukemogenic potential of a transformation-deficient BCR/ABL mutant

The oncogenic BCR/ABL protein protects hematopoietic cells from apoptosis induced by growth factor deprivation, but the mechanisms are only partially understood. A BCR/ABL mutant lacking amino acids 176-426 in the BCR domain (p185DeltaBCR) failed to protect interleukin 3-deprived 32Dcl3 myeloid precursor cells from apoptosis, although it possessed tyrosine kinase activity and was capable of activating the Ras-Raf-MAP kinase pathway. Compared to p185 wild-type transfectants, p185DeltaBCR-transfected cells showed markedly reduced levels of Bcl-2 and expressed the hypophosphorylated, proapoptotic form of BAD. Bcl-2 expression in the mitochondrial fraction of p185DeltaBCR cells was also markedly diminished and mitochondrial RAF was undetectable. In p185DeltaBCR cells transfected with a mitochondria-targeted, constitutively active RAF (M-Raf) BAD was expressed in the hyperphosphorylated form and released from the mitochondria into the cytosol. p185DeltaBCR/M-Raf-transfected cells were completely resistant to apoptosis induced by growth factor deprivation in vitro. Moreover, constitutive expression of dominant-negative M-Raf (K375W) enhanced the susceptibility of 32Dcl3 cells expressing wild-type BCR/ABL to apoptosis. In severe combined immunodeficiency (SCID) mice, p185DeltaBCR/M-Raf double transfectants were leukemogenic, whereas cells expressing only p185DeltaBCR showed no leukemogenic potential. Together, these data support the existence of a BCR/ABL-dependent pathway that leads to expression of an active RAF in the mitochondria and promotes antiapoptotic and leukemia-inducing effects of BCR/ABL.     

Hinge bending within the cytokine receptor superfamily revealed by the 2.4 A crystal structure of the extracellular domain of rabbit tissue factor

Tissue factor (TF), a member of the cytokine receptor superfamily, is the obligate cofactor of coagulation factor VIIa (FVIIa), and has a pivotal role in initiating the extrinsic pathway of blood coagulation through formation of the TF x FVIIa complex. The crystal structure of the extracellular portion of rabbit TF has been solved at 2.35 A resolution and refined to a crystallographic R-value of 19.1% (free R-value, 27.7%). Like the human homologue, the extracellular portion consists of two fibronectin type III domains connected by a short alpha-helical segment. Unexpectedly, the two molecules in the crystallographic asymmetric unit differ in their relative domain-domain orientation, revealing unsuspected hinge motion consisting of a rotation of about 12.7 degrees around an axis intersecting the linker segment at residue 106. Superposition of rabbit tissue factor with free and bound human tissue factor allows for the detection of an identical, albeit smaller, hinge motion in human TF induced upon binding of FVIIa. This raises the possibility that a very similar hinge axis may be present in other members of the cytokine receptor superfamily.     

Renal resistive index in experimental partial and complete ureteral obstruction

RATIONALE AND OBJECTIVES: Recent clinical work suggests that the Doppler resistive index (RI) may be useful in distinguishing obstructive from nonobstructive hydronephrosis. We evaluated the usefulness of the RI in a rabbit model of hydronephrosis. METHODS: Unilateral partial ureteral obstruction was produced in nine rabbits and complete obstruction in another nine. Three sham operations were performed, and these animals served as control subjects. The RI was measured in all kidneys before and 6 hr after surgery and on days 1, 4, and 7 postoperatively. The RI and the difference in RI (delta RI) between the obstructed and normal kidney were evaluated over time using a two-way analysis of variance. The intravenous urography and Whitaker tests served as gold standards. RESULTS: Hydronephrosis was observed on sonograms in all obstructed kidneys. Comparing groups, there was no significant difference in mean RI or delta RI between the three groups at any time point. Looking at individual groups over time, there was no significant change in mean delta RI, whereas the change in mean RI was significantly elevated above baseline only in the complete obstruction group at 6 hr (p = .002) and on days 4 (p = .008) and 7 (p = .006). In evaluating varying thresholds of RI and delta RI, we could not consistently discriminate between normal and obstructed kidneys. CONCLUSION: Although complete obstruction caused a significant increase in RI, partial obstruction failed to do so. RI and delta RI values proved to be insensitive predictors of obstruction in this rabbit model.     

Natural infection of a homozygous delta24 CCR5 red-capped mangabey with an R2b-tropic simian immunodeficiency virus

A homozygous 24-bp deletion (Delta24) was found in the CC chemokine receptor 5 (CCR5) of 11 out of 15 red-capped mangabeys (RCMs), Cercocebus torquatus torquatus, both in Africa and in an American zoo. The CCR5 Delta24 defect encompassed eight amino acids in frame in the fourth transmembrane region. Unexpectedly, RCM-009, one of 11 homozygotes (Delta24CCR5/ Delta24CCR5), was found to be naturally infected with a divergent simian immunodeficiency virus (SIV) strain, which was not R5-tropic, but used CCR2b (R2b) as its major coreceptor. SIVrcmGab1 was the only R2b-tropic SIV among other divergent SIVs tested. Cells transfected with the Delta24 CCR5 did not support entry of R5-tropic SIVmac, SIVcpz, SIVmne, HIV-2, or HIV-1, and were also inactive in signal transduction mediated by beta-chemokines. At 86.6%, the Delta24 allelic frequency was significantly higher than that of the 32-bp deletion found in humans. The Delta24 frequency was 4.1% in 34 sooty mangabeys (SMs), a geographically isolated subspecies that was naturally infected with R5-tropic SIV. Finding identical deletions in two mangabey subspecies separated for 10,000 years or more dates the Delta24 CCR5 deletion as ancient. However, the source of the selective pressure for the high rate of CCR5 deletion in RCMs remains to be determined. The high allelic frequency of the Delta24 CCR5 in RCMs, in comparison to that of SMs, suggests that R2b-tropism may have been acquired by SIVrcm, as an adaptation to CCR5 genetic defects appeared in its host.     

Localization of the gene for rapidly progressive autosomal dominant parkinsonism and dementia with pallido-ponto-nigral degeneration to chromosome 17q21

The distribution of calcyclin in some chicken tissues was studied by Western blotting using polyclonal antibodies raised against calcyclin purified from chicken gizzard. The protein was found in gizzard muscle and in a lesser amount in skeletal and cardiac muscle. No immunological reaction was observed in chicken liver. Immunohistochemical studies of chicken gizzard tissue revealed the presence of calcyclin only in muscle fibers. Ca(2+)-dependent interaction of chicken gizzard calcyclin with potential protein targets was also examined. By gel overlay method it was found that calcyclin bound to three proteins with molecular masses of approximately 35 kDa, 25 kDa and 15 kDa present in the cytosolic fraction derived from chicken gizzard muscle. The chicken gizzard calcyclin was also shown to interact with lysozyme.     

Biomechanical evaluation of the medial collateral ligament of the elbow

Anatomical dissection and biomechanical testing were used to study twenty-eight cadaveric elbows in order to determine the role of the medial collateral ligament under valgus loading. The medial collateral ligament was composed of anterior, posterior, and occasionally transverse bundles. The anterior bundle was, in turn, composed of anterior and posterior bands that tightened in reciprocal fashion as the elbow was flexed and extended. Sequential cutting of the ligament was performed while rotation caused by valgus torque was measured. The anterior band of the anterior bundle was the primary restraint to valgus rotation at 30, 60, and 90 degrees of flexion and was a co-primary restraint at 120 degrees of flexion. The posterior band of the anterior bundle was a co-primary restraint at 120 degrees of flexion and a secondary restraint at 30 and 90 degrees of flexion. The posterior bundle was a secondary restraint at 30 degrees only. The reciprocal anterior and posterior bands have distinct biomechanical roles and theoretically may be injured separately. The anterior band was more vulnerable to valgus overload when the elbow was extended, whereas the posterior band was more vulnerable when the elbow was flexed. The posterior bundle was not vulnerable to valgus overload unless the anterior bundle was completely disrupted. The intact elbows rotated a mean of 3.6 degrees between the neutral position and the two-newton-meter valgus torque position. Cutting of the entire anterior bundle caused an additional 3.2 degrees of rotation at 90 degrees of flexion, where the effect was greatest. CLINICAL RELEVANCE: Physical findings in a patient who has an injury of the anterior bundle may be subtle, and an examination should be performed with the elbow in 90 degrees of flexion for greatest sensitivity. As the anterior bundle is the major restraint to valgus rotation, reconstructive procedures should focus on anatomical reproduction of that structure. Parallel limbs of tendon graft placed from the inferior aspect of the medial epicondyle to the area of the sublimis tubercle will simulate the reciprocal bands of the anterior bundle. Temporary immobilization with the elbow in flexion may relax the critically important anterior band of the reconstruction during healing.     

Differences in depression and self-esteem reported by learning disabled and behavior disordered middle school students

We investigated the clinical and angiographic risk profile of slow flow during rotational atherectomy. Lesion length, angina at rest, and use of beta blockers correlated independently with slow flow in the univariate as well as in the multivariate analysis.     

Second-order motion perception in peripheral vision: limits of early filtering

Spatial and temporal analysis of contrast-modulated sine-wave gratings reveals that the second-order motion stimulus contains two sidebands, with equal energy but moving in opposite directions, flanking a stationary carrier. Any early linear spatial filtering process in the visual system that attenuates one sideband more than the other will be detrimental to the balance between the two sidebands, so that the perceived direction of the carrier might be opposite to that of the envelope motion. We tested this hypothesis by using contrast-modulated gratings presented centrally or at 20 deg in the horizontal nasal field with a two-alternative forced-choice staircase paradigm. We found that when the envelope frequency was close to that of the carrier, a second-order stimulus whose envelope motion direction was correctly identified in the fovea appeared to drift in the opposite direction in the periphery. Further increasing the envelope spatial frequency resulted in a reversed motion percept in both central and peripheral viewing conditions. For subjects to identify correctly the direction of motion of the envelope, the spatial frequency ratio of the carrier to the envelope had to be more than 2 in the fovea and more than 6 in the periphery. These phenomena in second-order motion perception can be explained by a linear model of motion detection with an early spatial filtering process. Further experiments and computer simulation show that undersampling of the carrier has little effect on second-order motion perception in the periphery, as long as the carrier is detectable.     

The placental transfer of sufentanil: effects of fetal pH, protein binding, and sufentanil concentration

This study investigated factors that influence the placental transfer of sufentanil using the dual-perfused, single-cotyledon human placental model. Placentas were collected from healthy women. Experiments were designed to elucidate the effects of maternal protein binding, changing maternal sufentanil concentration (1, 10, 20, and 100 ng/mL) and decreasing fetal pH (fetal acidemia 7.2, 7.0, 6.8) on the placental transfer of sufentanil. Sufentanil crossed the placenta rapidly at a rate two-thirds that of the transfer marker, antipyrine. Sufentanil transfer increased linearly with the maternal concentration (r = 0.999). Sufentanil/antipyrine maternal to fetal (M-->F) transfer ratios were significantly reduced (0.66 +/- 0.05 vs 0.40 +/- 0.04, P < 0.05) when fresh frozen plasma was added to the maternal circuit to enhance protein binding. Fetal pH and sufentanil transfer were related because sufentanil M-->F clearance increased significantly as the fetal pH decreased (r = 0.973, P < 0.05). Sufentanil appears to cross the placenta by passive diffusion but is modulated by the degree of maternal protein binding. Sufentanil M-->F transfer is enhanced by fetal acidemia.