In vivo stimulation of I kappa B phosphorylation is not sufficient to activate NF-kappa B.

NF-kappa B is a major inducible transcription factor in many immune and inflammatory reactions. Its activation involves the dissociation of the inhibitory subunit I kappa B from cytoplasmic NF-kappa B/Rel complexes, following which the Rel proteins are translocated to the nucleus, where they bind to DNA and activate transcription. Phosphorylation of I kappa B in cell-free experiments results in its inactivation and release from the Rel complex, but in vivo NF-kappa B activation is associated with I kappa B degradation. In vivo phosphorylation of I kappa B alpha was demonstrated in several recent studies, but its role is unknown. Our study shows that the T-cell activation results in rapid phosphorylation of I kappa B alpha and that this event is a physiological one, dependent on appropriate lymphocyte costimulation. Inducible I kappa B alpha phosphorylation was abolished by several distinct NF-kappa B blocking reagents, suggesting that it plays an essential role in the activation process. However, the in vivo induction of I kappa B alpha phosphorylation did not cause the inhibitory subunit to dissociate from the Rel complex. We identified several protease inhibitors which allow phosphorylation of I kappa B alpha but prevent its degradation upon cell stimulation, presumably through inhibition of the cytoplasmic proteasome. In the presence of these inhibitors, phosphorylated I kappa B alpha remained bound to the Rel complex in the cytoplasm for an extended period of time, whereas NF-kappa B activation was abolished. It appears that activation of NF-kappa B requires degradation of I kappa B alpha while it is a part of the Rel cytoplasmic complex, with inducible phosphorylation of the inhibitory subunit influencing the rate of degradation.

Chronic obstructive pulmonary disease: rehabilitation program with continuation on an outpatient basis.

A total of 187 patients with chronic obstructive pulmonary disease (COPD) were treated in a rehabilitation program, initially as inpatients and then scheduled to continue 6 months or more in an outpatient clinic. The mean age was 61 (range, 28-76 years). Changes shown by participants in the outpatient program were compared to those shown by the nonparticipants. For both groups, the mortality rates were similar to published figures, and were strongly related to the levels of forced expiratory volume/sec (FEV 1.0). The FEV 1.0 declined significantly within one year in both groups. Psychologic test scores were unchanged. There was a sharp increase in unemployment. Although rehabilitative therapy must be continued, high priority should be given to early detection of COPD in patients who have airway obstruction but are otherwise asymptomatic. Possibly, at that stage, the elimination of cigarette smoking may slow the process.

Costimulation requirement for AP-1 and NF-kappa B transcription factor activation in T cells.

The transcriptional activity of the IL-2 promoter requires T-cell costimulation delivered by the TCR and the auxiliary receptor CD28. Several transcription factors participate in IL-2 promoter activation, among which are AP-1-like factors and NF-kappa B. Protein phosphorylation has an important role in the regulation of these two factors: (1) it induces the transactivating capacity of the AP-1 protein c-Jun; and (2) it is involved in the release of the cytoplasmic inhibitor, I kappa B, from NF-kappa B, allowing translocation of the latter into the nucleus. We have recently shown that both phosphorylation processes require T-cell costimulation. Furthermore, in activated T cells, the kinetics of the two phosphorylation events are essentially similar. According to our results, however, the kinases responsible for the two processes are distinct entities. Whereas TPCK inhibits phosphorylation of I kappa B and, consequently, activation of NF-kappa B, it markedly enhances the activity of JNK, the MAP kinase-related kinase that phosphorylates the transactivation domain of c-Jun. We, therefore, propose the activation scheme presented in FIGURE 3 for T-cell costimulation. Costimulation results in the activation of a signaling pathway that leads to the simultaneous induction of the two transcription factors, AP-1 and NF-kappa B. Integration of the signals generated by TCR and CD28 engagement occurs along this pathway, which then bifurcates to induce I kappa B phosphorylation and NF-kappa B activation on the one hand, and JNK activation and c-Jun phosphorylation on the other. We are currently engaged in defining where the two signals integrate along the AP-1/NF-kappa B pathway.

[Humeral fracture caused by arm wrestling].

Humeral shaft fractures may occur as a result of arm wrestling ("Indian wrestling," "iron arm"), a contest in which the antagonists sit face-to-face, grip hands with their elbows on a table, and try to force the opponent's arm back. A case of this type is reported.

[Intramedullary nailing of mid-femoral fractures].

75 consecutive cases of mid-shaft femoral fractures were treated by open intramedullary nailing, using the solid, diamond-shaped Hansen-Street nail. The average age was 32.5 years; road accidents accounted for 43 cases; average operative time was 40 minutes. There were 2 systemic complications but no infections. 56 of the 75 were studied after an average of 5 years and 19 were followed for at least 6 months. At 4 months all fractures were united except 1. Average time to sound bone union was 4 months. There was no residual deformity in varus-valgus or in the sagittal plane. Malrotation was rare and did not exceed 10 degrees. There was shortening of 2 cm in 3 cases of comminuted fractures. All patients followed up returned to regular daily function. The Hansen-Street nail used in an open procedure is an accurate method of anatomic reduction. As opposed to close techniques, we found the procedure to be simple and quick, without undue exposure of the surgical team to radiation and without other complications, such as nerve damage due to traction. This device is indicated for use only in fractures of the middle third of the femoral shaft.

The use of the Hansen-Street intramedullary nail in midshaft fractures of the femur.

A retrospective study was conducted on 75 consecutive femoral midshaft fractures treated with intramedullary nailing using the solid-diamond-shape Hansen-Street nail with an open technique. The average age of patients was 32.5 years. Road accidents accounted for 43 cases. The average operative time was 40 minutes. There were two systemic complications. No infection occurred. Fifty-six cases could be gathered for this study, with an average follow-up of 5 years; 19 were followed for at least 6 months. At 4 months, all the fractures were united except one. The average time to sound bony union was 4 months. There was no residual deformity in varus-valgus or in the sagittal plane. Malrotation was rare and did not exceed 5 degrees. Shortening of 2 cm was found in 3 patients where the fracture was comminuted. All followed patients returned to regular daily function. The use of the Hansen-Street nail in an open procedure is an accurate method for anatomic reduction. As opposed to closed techniques, the procedure proved in our hands to be simple and quick, without undue exposure to radiation of the surgical team, and without such other complications as nerve complications due to traction. The procedure with this device is indicated for mid-third fractures of the femoral shaft only.

Detection and treatment of congenital hip dislocation in babies at risk in Western Galilee.

The orthopedic department of the Nahariya Government Hospital, together with the Mother and Child Health centers of the Ministry of Health, is aiming at early detection of congenital dislocation of the hip in Western Galilee. In suspected cases, the baby is referred to the orthopedic outpatient department where a thorough examination, usually including X-ray, is performed. One result of early detection and consequent early conservative treatment is that the number of babies requiring surgery dropped from 16.7 to 5% in recent years.

Osteoid osteoma in an ossicle of the big toe.

The authors describe an unusual case of osteoid osteoma in the foot and discuss its clinical characteristics.

Tuberculosis of the subdeltoid bursa. A case report.

The 10th reported case of tuberculosis of the subdeltoid bursa is described. A 45-year-old Caucasian woman presented with a 30-year clinical history of subdeltoid bursitis, with typical physical signs, but with normal X-rays of the shoulder-joint region and lungs. The diagnosis was made by needle aspiration and was later confirmed by bacteriologic and histological examination of the surgical specimen. Following surgery and chemotherapy, the patient has remained free of any active tuberculosis disease for the past five years.

Monoclonal antibodies specific to the alpha-subunit of the mast cell's Fc epsilon R block IgE binding and trigger histamine release.

In an attempt to block the interactions between IgE and its receptor on mast cells (Fc epsilon R), we have established anti-Fc epsilon R monoclonal antibodies (mAb) by fusion of myeloma cells with mouse splenocytes immunized with irradiated rat basophilic leukemia (RBL) cells. Two anti-Fc epsilon R mAb were obtained (denoted 4.7 and 5.14) that could specifically bind to RBL and mast cells. This binding could be inhibited by IgE. The mAb and their F(ab')2 fragments inhibited 125I-IgE binding to RBL cell and triggered cell degranulation. The Fab' fragments, on the other hand, could only inhibit IgE binding but did not stimulate cell degranulation. Furthermore, these monovalent fragments inhibited RBL and mast cell degranulation induced by IgE-antigen complexes both in vitro and in vivo in the passive cutaneous anaphylaxis reaction. The number of mAb 4.7 and 5.14 molecules bound per RBL cells was similar to that of IgE; nevertheless, mAb 4.7 and 5.14 recognized different epitopes on the IgE receptor. Immunoprecipitation and immunoblotting analysis demonstrated that the mAb reacted with the alpha-subunit of the Fc epsilon R. Our findings establish the anti-Fc epsilon R mAb as a useful reagent for the isolation and characterization of the Fc epsilon R's alpha-subunit and the monomeric (Fab') for blocking the IgE-Fc epsilon R interactions.

Redox regulation of a protein tyrosine kinase in the endoplasmic reticulum.

The subcellular localization of the mouse Ltk transmembrane protein tyrosine kinase was studied in transfected COS cells, a mature B lymphocyte line, and a low expressing transfected lymphocyte clone. Indirect immunofluorescence and immunogold staining of COS transfectants and endoglycosidase analysis of both COS transfectants and lymphocytes indicate the unusual localization of Ltk to the endoplasmic reticulum (ER). Ltk resembles a receptor tyrosine kinase; it has a short, glycosylated, and cysteine-rich N-terminal domain. Yet, it appears to function in a ligand-independent mechanism: its in vivo catalytic activity is markedly enhanced by alkylating and thiol-oxidizing agents, and the active fraction of the protein occurs as disulfide-linked multimers. The catalytic activity of Ltk in the ER may be regulated via changes in the cellular redox potential, a novel mechanism for regulating protein tyrosine kinases. The ability to respond to redox changes in the cell may, however, be shared with certain receptor kinases during their passage through the ER.

Pulmonary vascular changes following air embolism in the dog.

The effect of an intravenous injection of air in a dose of 1 ml/kg body weight was determined in 15 healthy mongrel dogs. In 4 control dogs the mean pulmonary artery pressure rose to 2-3 times the resting values at 30 seconds, and carbon monoxide diffusing capacity and pulmonary capillary blood volume decreased by half. In the animals pretreated either with heparin or with methysergide (antiserotonin group) the results were the same as in the control animals. In the vagotomized dogs, the rise in pulmonary artery pressure was not significant, and the decrease in pulmonary capillary blood volume was of lesser magnitude and shorter duration than in the control and the antiserotonin dogs. It is concluded that the intravenous injection of air in supine dogs causes a transient obstruction of small pulmonary arteries. Evidence is presented to implicate a vagal mechanism in both main aspects of the response, namely the pulmonary artery pressure rise, and the partial obstruction of the pulmonary capillary bed. These studies offer additional explanation of the symptoms of respiratory distress observed in rapid decompression.

IL-3 facilitates lymphocyte hexose transport by enhancing the intrinsic activity of the transport system.

Interleukin 3 (IL-3) promotes the survival and proliferation of hematopoietic cells of various lineages in culture. Like most other hematopoietic colony stimulating factors, its mode of action is unknown. However, binding of the lymphokine induces protein tyrosine phosphorylation and enhanced glucose transport in some myeloid progenitor cells. We have studied the hexose uptake following IL-3 stimulation in IL-3-dependent pro-B cells. IL-3 facilitated the uptake of 2-deoxyglucose within 15 min. Kinetic analysis of the 2-deoxyglucose uptake attributed the enhanced transport to improved transporter function, while other hormones or cytokines affect glucose transport primarily via the number of cell surface transporters.

Stimulation-dependent I kappa B alpha phosphorylation marks the NF-kappa B inhibitor for degradation via the ubiquitin-proteasome pathway.

The nuclear translocation of NF-kappa B follows the degradation of its inhibitor, I kappa B alpha, an event coupled with stimulation-dependent inhibitor phosphorylation. Prevention of the stimulation-dependent phosphorylation of I kappa B alpha, either by treating cells with various reagents or by mutagenesis of certain putative I kappa B alpha phosphorylation sites, abolishes the inducible degradation of I kappa B alpha. Yet, the mechanism coupling the stimulation-induced phosphorylation with the degradation has not been resolved. Recent reports suggest a role for the proteasome in I kappa B alpha degradation, but the mode of substrate recognition and the involvement of ubiquitin conjugation as a targeting signal have not been addressed. We show that of the two forms of I kappa B alpha recovered from stimulated cells in a complex with RelA and p50, only the newly phosphorylated form, pI kappa B alpha, is a substrate for an in vitro reconstituted ubiquitin-proteasome system. Proteolysis requires ATP, ubiquitin, a specific ubiquitin-conjugating enzyme, and other ubiquitin-proteasome components. In vivo, inducible I kappa B alpha degradation requires a functional ubiquitin-activating enzyme and is associated with the appearance of high molecular weight adducts of I kappa B alpha. Ubiquitin-mediated protein degradation may, therefore, constitute an integral step of a signal transduction process.

Rapid proteolysis of I kappa B-alpha is necessary for activation of transcription factor NF-kappa B.

Inducible gene expression in eukaryotes is mainly controlled by the activity of transcriptional activator proteins, such as NF-kappa B (refs 1-3), a factor activated upon treatment of cells with phorbol esters, lipopolysaccharide, interleukin-1 and tumour necrosis factor-alpha. Activation of NF-kappa B involves release of the inhibitory subunit I kappa B from a cytoplasmic complex with the DNA-binding subunits Rel-A (formerly p65) and p50 (refs 6, 7). Cell-free experiments have suggested that protein kinase C and other kinases transfer phosphoryl groups onto I kappa B causing release of I kappa B and subsequent activation of NF-kappa B. Here we report that I kappa B-alpha (formerly MAD-3) is degraded in cells after stimulation with phorbol ester, interleukin-1, lipopolysaccharide and tumour necrosis factor-alpha, an event coincident with the appearance of active NF-kappa B. Treatment of cells with various protease inhibitors or an antioxidant completely prevented the inducible decay of I kappa B-alpha as well as the activation of NF-kappa B. Our findings suggest that the activation of NF-kappa B relies on an inducible degradation of I kappa B-alpha through a cytoplasmic, chymotrypsin-like protease. In intact cells, phosphorylation of I kappa B-alpha is apparently not sufficient for activation of NF-kappa B.

Mapping of murine IgE epitopes involved in IgE-Fc epsilon receptor interactions.

The generation of anti-IgE monoclonal antibodies has permitted the identification of various serological epitopes on the IgE molecule. The relationship of the sites on IgE recognized by such antibodies to the Fc epsilon receptor (Fc epsilon R) interaction site has been determined using cross-inhibition studies. However, interpretation of this type of experiment is limited by problems of steric hindrance. Thus, to accomplish precise mapping on the IgE molecule of the Fc epsilon R interaction site and the binding sites of various anti-IgE mAb, we employed site-directed mutagenesis of the IgE heavy chain gene. To this end we have constructed and expressed a recombinant murine constant epsilon heavy chain (C epsilon) gene bearing a (4-hydroxy-3-nitrophenyl)acetic acid (NP)-binding VH region. Several site-specific mutants in the C epsilon 3 and C epsilon 4 domains of this recombinant C epsilon gene were prepared and expressed by transfection into the light chain-producing J558L myeloma cell line. The resulting IgE antibodies were tested for binding to mast cells and to various anti-IgE mAb. The mutants produced include a proline to histidine point mutant at amino acid residue 404 in the C epsilon 3 domain, a mutant with a truncated C epsilon 4 domain, a mutant with a 45 amino acid deletion in the carboxy end of C epsilon 3, and a chimeric human C epsilon in which the human C epsilon 3 was replaced by the homologous mouse C epsilon 3 domain. These mutants have permitted the localization, to the C epsilon 3 domain, of the epitopes recognized by the 84.1C and 95.3 anti-IgE mAb. The 84.1C mAb recognizes a site on IgE which is identical or very close to the Fc epsilon R binding site, and 95.3 recognizes a site on IgE which is related, but not identical to the Fc epsilon R binding site. The antigenic determinant recognized by the 51.3 mAb, which is inefficient at blocking the IgE-Fc epsilon R interaction, has been mapped to the C epsilon 4 domain. When tested for binding to the Fc epsilon R on RBL-2H3 cells, the point mutant bound to the Fc epsilon R with twofold reduced affinity, while the C epsilon 3 deletion mutant and the mutant truncated in C epsilon 4 lost all receptor binding activity.(ABSTRACT TRUNCATED AT 400 WORDS)