A systematic approach to the complete study of a signaling molecule: ribosomal p90rsk as an example.

Ribosomal p90rsk is a kinase of central importance in transducing mitogenic signals from an activated receptor to the cell nucleus and for protein synthesis. Here, we analyze the optimal steps to fully describe this kinase in both normal neutrophils and leukemic cell lines. These are: (i) immunological analyses (immunoblotting and immunoprecipitation); (ii) enzyme activity assays (in vitro and "in-gel"); and (iii) immunobiochemical combination methods (immunoprecipitation/kinase assay, immunoprecipitation/"in-gel" assay and ion exchange chromatography/immunoblotting). For the enzyme assays, we describe a novel method to measure ribosomal p90rsk kinase activity "in-gel", based on a renatured-protein method that allows for the direct quantitation of enzyme activity. Finally, we present an algorithm that can be readily implemented to the quantification of the extent of stimulation of a kinase in response to a particular extracellular stimuli. In our case, it was found that activation of p90rsk was higher in proliferating leukemic cells than in mature neutrophils, indicating that a suppression of key signal transduction links could contribute to the maturational arrest typical of acute leukemia. All the techniques and strategies described here for p90rsk could be easily extrapolated to the study of any signal transduction molecule, provided it has a phosphotransferase activity.

Presence of a phospholipase D (PLD) distinct from PLD1 or PLD2 in human neutrophils: immunobiochemical characterization and initial purification.

Utilizing the transphosphatidylation reaction catalyzed by phospholipase D (PLD) in the presence of a primary alcohol and the short-chain phospholipid PC8, we have characterized the enzyme from human neutrophils. A pH optimum of 7.8-8.0 was determined. PIP(2), EDTA/EGTA, and ATP were found to enhance basal PLD activity in vitro. Inhibitory elements were: oleate, Triton X-100, n-octyl-beta-glucopyranoside, divalent cations, GTPgammaS and H(2)O(2). The apparent K(m) for the butanol substrate was 0.1 mM and the V(max) was 6.0 nmol mg(-1) h(-1). Immunochemical analysis by anti-pan PLD antibodies revealed a neutrophil PLD of approximately 90 kDa and other bands recognized minimally by anti-PLD1 or anti-PLD2 antibodies. The 90-kDa protein is tyrosine-phosphorylated upon cell stimulation with GM-CSF and formyl-Met-Leu-Phe. Protein partial purification using column liquid chromatography was performed after cell subfractionation. Based on the enzyme's regulatory and inhibitory factors, and its molecular weight, these data indicate an enzyme isoform that might be different from the mammalian PLD1/2 forms described earlier. The present results lay the foundation for further purification of this granulocyte PLD isoform.

An outcomes analysis and satisfaction survey of 199 consecutive abdominoplasties.

Abdominoplasty is a popular body-contouring procedure. In this study the authors review retrospectively 199 abdominoplasty patients during a 15-year period to identify factors that affect overall outcome. Patients included 190 women and 9 men. The complication rate was 32% with few major complications (1.4%). The revision rate was 43%, and was related to fine-tuning the aesthetic appearance. Patients were divided into four groups based on tobacco use and history of diabetes and hypertension. There was no significant difference in revision rates or major complications between the subgroups. Minor complication rates, however, were significantly higher in smokers and patients with diabetes and/or hypertension. Complication and revision rates in patients undergoing intra-abdominal procedures combined with abdominoplasty were not significantly different from those patients undergoing abdominoplasty alone. A patient survey revealed symptom improvement in 95% of patients. Eighty-six percent of patients were satisfied with their result, and 86% would recommend abdominoplasty to a friend. The authors conclude that abdominoplasty is a safe and satisfying procedure, whether performed alone or in conjunction with another procedure. Patients are pleased with the outcome and have improvement in their symptoms, with minimal health risk. There is, however, a significant incidence of minor complications, related primarily to wound healing. These complications are increased significantly in smokers and patients with diabetes and/or hypertension. Revision rates are not different significantly between the subgroups. When complications do occur or revisions are required, they are minor and managed easily in an office setting.

MAP kinase upregulation after hematopoietic differentiation: role of chemotaxis.

Mitogen-activated protein kinase (MAPK) isoform p42 is known to be active in exponentially growing cells at several points of the cell cycle. A high basal activity was present in three cell lines representative of immature myeloid cells tested: uHL-60, AML-14, and MPD. However, DMSO-induced differentiation of HL-60 cells (dHL-60) and subsequent expression of the neutrophilic phenotype occurred with a concomitant reduction on the basal level of MAPK activity. Simultaneously, extracellular stimuli like the cytokine granulocyte/macrophage colony-stimulating factor (GM-CSF) induced a fast (<10 min) and robust response. In terms of MAPK activity, the more mature the cell was, the higher the corresponding activity, in the three differentiation series considered: AML-14 < 3D10; MPD < G-MPD; uHL-60 < dHL-60 < neutrophils. Interestingly, peripheral blood neutrophils expressed the highest (16-fold) MAPK activation level in response to GM-CSF. Finally, using the specific MAPK inhibitor PD-98059, we demonstrated that MAPK activation is needed for neutrophil chemotaxis toward interleukin-8 and its priming by GM-CSF. Since neutrophils are terminally differentiated cells, GM-CSF does not serve a purpose in proliferation, and it must trigger the recruitment of selective signal transduction pathways particular to that final stage that includes enhanced physiological functions such as chemotaxis.

Human cell line that differentiates to all myeloid lineages and expresses neutrophil secondary granule genes.

The aim of this study was to characterize a human leukemic cell line that appears capable of spontaneous differentiation to all myeloid lineages. The MPD cell line was derived using standard tissue culture techniques from the peripheral blood of a patient with an aggressive nonchronic myelogenous leukemia myeloproliferative disorder. Immunophenotyping, cytogenetic analysis, reverse transcriptase polymerase chain reaction, Northern blotting, immunoblotting, and colony assays were used to characterize the line and to assess its ability to express lineage-specific genes representative of advanced differentiation.Light microscopic morphologic analysis of the MPD cell line suggests that it has the unique property of spontaneous differentiation to mature-appearing neutrophils, macrophages, eosinophils, and basophils in proportions that approximate those found in normal bone marrow or peripheral blood. It was demonstrated that this cell line is capable of producing lineage-specific mRNA and granule proteins of at least two myeloid lineages, neutrophil and eosinophil, including neutrophil secondary granule proteins, which are not expressed in other available human cell lines. MPD cells were found to be capable of producing differentiated myeloid colonies (neutrophil, eosinophil, macrophge, mixed) in semisolid medium. The ability of MPD cells to express genetic programs associated with advanced differentiation of multiple myeloid lineages will make it a valuable tool for the study of the processes underlying lineage commitment and the regulation of expression of lineage-specific genes.

Lung herniation into the neck associated with congenital absence of the sternocleidomastoid muscle.

This is a case report and literature review involving congenital absence of the sternocleidomastoid muscle (SCM) and simultaneous herniation of the left lung into the neck. To date there have been eight cases of absence of the SCM muscle, but no records of lung herniation alone or in combination with SCM muscle absence. Included is the management course taken to correct this patient's defect, CAT scans of the neck, fluoroscopic imaging of the lung and neck, and photographic documentation of the defect.

Reduction mammaplasty: an outcome analysis.

Reduction mammaplasty is usually performed to relieve painful symptoms and physical signs of macromastia. Justification for reduction mammaplasty should be based on the probability of relieving these clinical signs and symptoms. This retrospective study involved four surgeons who performed a variety of breast reduction procedures and was designed to determine if preoperative symptoms were resolved after reduction mammaplasty. We surveyed 285 women who had reduction mammaplasties from 1988 to 1993. Data from these surveys and the patients' charts were reviewed. A total of 185 patients (65%) returned completed surveys and were included in this study for analysis. Mean age was 40 years with an average follow-up of 3 years. The most common preoperative complaint was shoulder grooving (90%), followed by back pain (82%), shoulder pain (78%), and neck pain (65%). Average amount of breast tissue removed was 855 gm from each breast. Preoperative complaints were substantially reduced after surgery, regardless of the presurgical body mass. Most patients (97%) had improvement of symptoms, and 59% were asymptomatic. Only 3% had no change in their symptoms and none were worse. The complication rate was 45% with fat necrosis/infection being the most common complication (22%). The majority of patients (95%) were either happy or very happy with the surgery, and 98% would recommend surgery to a friend. Our data indicate that reduction mammaplasty relieves preoperative symptoms associated with macromastia.

Orthognathic surgery in the cleft lip and palate patient.

Orthognathic surgery for the cleft lip and palate patient should be designed to achieve good facial aesthetics and a stable, functional occlusion. Maxillary and mandibular osteotomies, which benefit cleft lip and palate patients with associated dentofacial deformities, should be modified to meet the needs of the individual patient. Soft-tissue correction of the upper lip and nose adds to the overall aesthetic result, but should be performed as a separate procedure.

Comparison of Nasometer and listener judgments of nasality in the assessment of velopharyngeal function after pharyngeal flap surgery.

This study examined the relationship between nasalance scores as derived from the Model 6200 Nasometer and listener judgments of perceived nasality for individuals with pharyngeal flaps. Sixteen individuals with pharyngeal flaps read a speech sample consisting of seven sentences for which associated nasalance scores were obtained. In addition, 10 trained listeners were asked to judge the subjects' audiorecorded speech samples for the degree of both hypernasality and hyponasality using two 6-point scales. The mean judges' ratings of hypernasality did not increase systematically with increasing nasalance scores or with decreasing hyponasality ratings. However, as the nasalance scores associated with nasal loaded sentences increased, a systematic decrease in listener perception of hyponasality occurred.

One-stage closure of the entire primary palate.

Timing of the closure of the anterior palate and alveolus is a subject of debate. Late repair of this defect is complicated by high fistula formation and subjects the patient to the problems of palate fistula for extended periods of time. We have utilized a single procedure performed when the child is 3 months of age that completely closes the anterior hard palate and alveolus along with the cleft lip. Our series consisted of 61 consecutive patients with unilateral clefts of the primary and secondary palate. Mucosal turnover flaps from the vomer along with lateral nasal mucosal flaps provide the nasal lining. A buccal sulcus flap with a Veau flap completes the oral repair. Ninety-five percent (58 of 61) of the patients had complete and stable closure of their anterior palate and alveolus after 1 year. The incidence of fistula formation in our series (3 of 61) is much lower than that reported with the utilization of other protocols. Excellent exposure of the anterior palate and alveolar defect during lip repair, early restoration of anatomic relationships, establishment of a good nostril floor and sill, and very low fistula formation are among the benefits of this procedure. The increase in operative time is considered minimal in light of aforementioned advantages.

Surgical-orthodontic correction of transverse maxillary deficiency.

A conservative osteotomy of the zygomaticomaxillary buttress in combination with a rapid palatal expansion appliance is a dependable technique for the treatment of horizontal maxillary deficiency in adults. This procedure has been used successfully in 56 patients in our series, but 17 patients (30 per cent) required a midpalatal osteotomy. In two patients, overexpansion was not achieved because of necrosis of the mucosa. In three other patients, expansion had to proceed at a slower pace because of mucosal ulceration. There have been no other complications. The procedure is indicated mainly in those patients with a horizontal deficiency who do not require subsequent surgery, but for some patients it may be the preliminary procedure. Twelve patients (21 per cent) had subsequent orthognathic surgery. Follow-up has been from 1 to 12 years and there has been no relapse. In our opinion, the zygomaticomaxillary buttress is the primary area of resistance to lateral movement of the maxilla by rapid maxillary expansion appliances.

Surgical-orthodontic correction of transverse maxillary deficiency.

A conservative osteotomy of the zygomaticomaxillary buttress in combination with a rapid palatal expansion appliance is a dependable technique for the treatment of horizontal maxillary deficiency in adults. This procedure has been used successfully in 56 patients in our series, but 17 patients (30 percent) required a midpalatal osteotomy. In two patients, overexpansion was not achieved because of necrosis of the mucosa. In three other patients, expansion had to proceed at a slower pace because of mucosal ulceration. There have been no other complications. The procedure is indicated mainly in those patients with a horizontal deficiency who do not require subsequent surgery, but for some patients it may be the preliminary procedure. Twelve patients (21 per cent) had subsequent orthognathic surgery. Follow-up has been from 1 to 12 years and there has been no relapse. In our opinion, the zygomaticomaxillary buttress is the primary area of resistance to lateral movement of the maxilla by rapid maxillary expansion appliances.

Mandibular prognathism.

The use of the vertical subcondylar osteotomy for correction of uncomplicated mandibular prognathism is reviewed. Techniques for use in more complicated cases such as when a set back of greater than 10 mm is required are also described.

Soft-tissue manifestations of aesthetic defects of the jaws: diagnosis and treatment.

Treatment planning for patients with dentofacial deformities should include a skeletal and a soft-tissue analysis. This article collates and summarizes several different soft-tissue analyses. In addition, it correlates the anticipated soft-tissue profile changes with various skeletal and soft-tissue surgical procedures.

Surgical orthodontic correction of transverse maxillary deficiency: a simplified approach.

Osteotomy of the zygomaticomaxillary buttress in combination with a rapid palatal exapnsion appliance is a dependable technique for the treatment of horizontal maxillary deficiency in adults. This procedure has been used in 18 patients with excellent expansion in 17. In one patient, expansion was discontinued prior to overcorrection because of pressure necrosis on the palate related to the appliance. This was the only complication. The procedure is indicated mainly in those patients with a horizontal deficiency who do not require subsequent surgery, but for some patients it may be the preliminary procedure. Three patients in this series had subsequent orthognathic surgery. Follow-up has been from 1 to 6 years, and there has been no relapse. In our opinion, the zygomaticomaxillary buttress is the primary area of resistance to lateral movement of the maxilla by rapid maxillary expansion appliances.

The combined surgical and orthodontic treatment of mandibular prognathism.

Patients with severe mandibular prognathism are best managed with a combined orthodontic-surgical approach. In our patients, the orthodontic treatment consisted of six to eighteen months of presurgical preparation, which in some patients may accentuate the dental deformity. This is done to provide two well-aligned dental arches that will fit accurately at surgery. The surgical procedure used was an oblique subcondylar osteotomy. This was followed by six to eight months of orthodontic treatment to complete dental alignment. Thirty patients were treated using this combined approach, with excellent results and few complications.