Maintenance of oxidative phosphorylation protects cells from Actinobacillus actinomycetemcomitans leukotoxin-induced apoptosis.

Subnanomolar concentrations (3 x 10(-10) M) of Actinobacillus actinomycetemcomitans leukotoxin (Ltx) trigger apoptosis of JY cells, as shown by a decrease in mitochondrial transmembrane potential (DeltaPsim), hyperproduction of reactive oxygen species (ROS) and release of cytochrome c from the intermembrane space. When compared with heat-inactivated leukotoxin (DeltaI Ltx) controls, ATP levels in Ltx-treated JY cells continued to decrease during a 24 h experiment while cytoplasmic ADP concentrations were increasing. These results suggest that a blockage occurred in ATP/ADP exchange. To maintain ATP/ADP exchange, JY cells were transfected with bcl-2 and bcl-xL and incubated with Ltx. ATP levels of the transfected cells decreased to 67% (JY/bcl-2) and 73% (JY/bcl-xL) after the experiment. Furthermore, cytochrome c remained localized to the mitochondrial fraction of Ltx-treated JY/bcl-2 and JY/bcl-xL cells, whereas its presence in the cytoplasmic fraction of JY/gen cells suggests an uncoupling of electron transport. Expression of bcl-2 and bcl-xL in cells inhibited downstream apoptotic events such as cleavage of poly(ADP-ribose) polymerase, DNA fragmentation and activation of a family of caspases. The results indicate that Ltx induces apoptosis through a mitochondrial pathway that involves decreased levels of the ADP in the mitochondrial matrix, a lack of substrate for ATP synthetase and arrest of oxidative phosphorylation.

Extracellular matrix and nuclear localization of beta ig-h3 in human bladder smooth muscle and fibroblast cells.

The extracellular matrix (ECM) plays an essential role in bladder structure and function. In this study, expression of beta ig-h3, a recently identified extracellular matrix protein, was investigated in human bladder tissue, and human bladder smooth-muscle (SMC) and fibroblast cells in vitro. SMCs secreted greater than three times the level of this protein compared with fibroblasts. The relative levels of beta ig-h3 mRNA in the two cell types reflected the protein expression. Immunohistochemical analysis demonstrated protein deposition in the ECM as well as cytoplasmic localization and, unexpectedly, nuclei. Anti-beta ig-h3 antibodies also stained the matrix surrounding the detrusor SMCs and nuclei of bladder fibroblasts, SMCs, and urothelium in intact bladder tissue. Western blot analyses of medium and matrix fractions obtained from cells in vitro revealed protein of approximately 70-74 kDa, whereas nuclear extracts contained a 65-kDa reactive protein band. We propose that although this protein is a structural component of bladder ECM, its nuclear localization suggests that it has other regulatory and/or structural functions.

Expression of betaig-h3 by human bronchial smooth muscle cells: localization To the extracellular matrix and nucleus.

Bronchial smooth muscle cells play a central role in normal lung physiology by controlling airway tone. In addition, airway smooth muscle hyperplasia and hypertrophy are important factors in the pathophysiology of asthma. In this study, expression of betaig-h3, a recently identified component of the extracellular matrix (ECM), was investigated in primary human bronchial smooth muscle (HBSM) cells. Northern blot analysis demonstrated that treatment of cultured HBSM cells with transforming growth factor-beta1 resulted in a 4- to 5-fold increase in the steady-state level of betaig-h3 messenger RNA. Western blot analysis of secreted proteins using monospecific antibodies generated against peptide sequences found in the N- and C-terminal regions of the protein identified several isoforms having apparent mass of 70-74 kD. Immunohistochemical analysis of human lung localized betaig-h3 to the vascular and airway ECM, and particularly to the septal tips of alveolar ducts and alveoli, suggesting that it may have a morphogenetic role. Analysis of cultured HBSM cells identified betaig-h3 in both the ECM as well as the cytoplasm, and surprisingly also in the nucleus. These results demonstrate that betaig-h3 is produced by resident lung cells, is a component of lung ECM, and may play an important role in lung structure and function. The presence of this protein in nuclei suggests that it may have regulatory functions in addition to its role as a structural component of lung ECM.

The role of endoscopy and implant texture in transaxillary submuscular breast augmentation.

In 1996 the authors reported their experience with 92 consecutive patients undergoing traditional (nonendoscopic) transaxillary submuscular breast augmentation. They reported a capsular contraction rate of 1.1% using textured saline implants, as well as an implant malposition rate of 8.6%. At that time they hypothesized that "the endoscope will lessen the rate of implant malposition." To clarify the role of implant texture and to justify the use of endoscopic techniques in transaxillary submuscular breast augmentation, the authors have studied an additional 58 consecutive patients with two significant changes. First, the endoscope was used to dissect the implant pocket in each patient. Second, smooth-wall implants were substituted for the previously used textured implants. Their rate of implant malposition dropped significantly, from 8.6% to 2.0% (p = 0.10), and their capsular contraction rate remained low, at 2.0% (p = 0.63). They conclude that direct endoscopic control improves implant positioning in the transaxillary submuscular operation, and implant texture does not improve capsular contraction rates when the prosthesis is placed in the submuscular position.

Molecular analysis of collagens in bladder fibrosis.

PURPOSE: Fibrosis of bladder tissue is characterized by an abnormal deposition of connective tissue within different layers of the bladder wall, resulting in a low volume, high pressure vesical which may ultimately contribute to renal scarring and failure. These bladders are functionally referred to as "non-compliant" and may result from different etiologies: neurogenic, which encompasses myelodysplasia and spinal cord injury, or non-neurogenic, owing to obstruction or radiation therapy. To examine the molecular mechanisms responsible for this fibrosis, we have analyzed a well-characterized pediatric patient population for alteration(s) in collagen types I and III regulation at the protein and nucleic acid levels. MATERIALS AND METHODS: Immunohistochemical localization of collagen subtypes (I, III, and IV) was carried out using type specific monoclonal antibodies. Total collagen was determined by hydroxyproline analysis, and subtype specific expression of collagenous proteins, following cyanogen bromide extraction procedures, was quantified by competitive ELISA. Total RNA was extracted by guanidinium/phenol/chloroform, and slot blot hybridization analyses with radiolabeled human cDNA probes were quantified by densitometry of resulting autoradiograms. RESULTS: Connective tissue infiltration of detrusor smooth muscle bundles was specific for type III collagen. Protein analyses demonstrated: 1) an increase in total collagen, 2) a statistically significant increase in the type III: type I collagen ratio, and 3), an absolute increase in type III collagen protein in non-compliant bladder tissue. At the mRNA level, there was a coordinate increase in both collagen I and III steady-state mRNAs in non-neurogenic bladder tissue, whereas neurogenic bladder tissue was characterized by an increase in the type III: type I mRNA transcript ratio. CONCLUSIONS: These data suggest that regulation of collagen synthesis in bladder fibrosis is complex and is characterized by both transcriptional and post-transcriptional mechanisms, depending upon the etiology of the fibrosis.

A modification of the primary nasal correction in the rotation-advancement unilateral cleft lip repair.

Primary correction of the unilateral cleft lip nasal deformity remains a challenge to the cleft surgeon. We wish to present a method of primary nasal correction utilizing presurgical orthopedics with the Latham dentomaxillary advancement appliance, concomitant rotation-advancement cleft lip repair, gingivoperiosteoplasty, and immediate correction of the nasal cartilage distortion with an "open technique" utilizing the rotation-advancement incisions. Direct elevation and suture fixation of the cleft side alar cartilage is performed as well as recreation of the normal upper lateral/lower lateral cartilage relationship, repair of nasal web, and release of the vestibular lining utilizing an L-flap pedicled to the alar base. Details of the operative technique, observations of unilateral cleft nasal anatomy as visualized with the "open technique," and preliminary results are illustrated in patients followed as long as 5 years.

Soft-tissue coverage of the neural elements after myelomeningocele repair.

We retrospectively reviewed all newborns with a diagnosis of myelomeningocele (MMC) admitted to our hospital between January 1990 and September 1994 to determine methods of soft tissue coverage, complication rates, and results. Sixty-five patients underwent repair of thoracic, lumbar, or sacral MMCs. The average size of defect repaired measured 21.3 cm2 (range, 2-80 cm2). Methods of repair included direct approximation of soft tissues with or without undermining (N = 48), Romberg Limberg flaps (N = 8), gluteus maximus or latissimus dorsi musculocutaneous flaps (N = 5), fascioutaneous flaps (N = 3), and V-gamma advancement (N = 1). A total of 18 complications were recorded (27.7%). There were 5 major complications (7.7%) and 13 minor ones (20.0%). Major complications were defined as midline wound dehiscence overlying the neural elements or wound infection leading to meningitis or ventriculitis. All 5 major and 9 minor complications arose in patients undergoing direct soft-tissue approximation. Additionally, all major complications were recorded in defects > 18 cm2. Based on this series, it appears that MMC defects < 18 cm2 can be closed by direct approximation of soft tissues without significant risk or major wound complication. Larger wounds may be successfully closed in this manner, but the risk of major complication is substantial.

Endoscopic transaxillary submuscular augmentation mammaplasty with textured saline breast implants.

The utility of the endoscope was realized in a retrospective analysis of 92 consecutive patients undergoing augmentation mammaplasty with saline-filled textured implants. The transaxillary submuscular approach was used. All patients received preoperative antibiotics and instillation of methylprednisolone into the saline fill, with inflation to manufacturer-recommended levels. complications included seven implant deflations (3.8%), eight implant malpositions (4.3%), and one capsular contracture (0.6%). Of the eight malpositions, six were corrected using the endoscopic technique without removal of the implant and with early manipulation of the implant within the pocket. We conclude that endoscopic control in augmentation mammaplasty is beneficial in secondary operative procedures and we speculate that the endoscope will lessen the rate of implant malposition observed in this study.

Complications in endoscopic plastic surgery.

Owing to the relatively short time endoscopic-assisted plastic surgery procedures have been done, there are few published reports of complications. The experiences of the faculty from the Endoscopy in Plastic Surgery: A consensus Multidisciplinary Symposium as well as the few published reports in the literature have been reviewed. The complications associated with endoscopic techniques are similar to those with open techniques. It is clear, however, that a subset of complications specific to endoscopic procedures exists. As endoscopic techniques and instrumentation are further developed, and as surgeons move higher up on the "learning curve", these complications should be reduced.

Minimal expansion for correction of male pattern baldness.

Tissue expansion for correction of male pattern baldness has not gained wide acceptance by patients or surgeons because of the substantial albeit temporary deformity of the expansion. Minimal expansion is an alternative. Scalp flaps can be expanded just to the point of becoming noticeable over 4 to 6 weeks followed by scalp flap transposition and easy closure of the donor site. The temporoparietal (Juri) or superiorly based (Dardour) flaps have been used unilaterally or bilaterally. The entire restoration is completed in approximately 8 weeks, is minimally deforming during the expansion phase, and does not require the patient to alter his lifestyle significantly or to go into hiding. Hair grafts, in contrast, do not begin to grow hair until 12 weeks after transplantation, and the process usually requires four sessions over an 8- to 12-month period with the patient looking temporarily variably absurd. Tissue expansion increases scalp available for flap restorations, which appear more natural than even the most well-executed hair transplants. Minimal expansion makes the process more palatable to the patient and surgeon.

Sequencing of the primary adhesion domain of bovine von Willebrand factor.

A cDNA library, constructed from bovine heart endothelial cell poly(A)+ RNA, was screened using a BstXI fragment of human von Willebrand and factor (vWF) cDNA as a probe. This probe codes for the major adhesion domain of vWF that includes the GPIb, collagen and heparin binding domains. Of the ten positive clones obtained, a clone that spanned the region of interest was sequenced by the dideoxynucleotide method yielding a sequence of 1550 bp. This region of the bovine cDNA codes for amino acids corresponding to #262 to #777 in human vWF and encompasses the entire pro adhesion domain. Both the nucleotide sequence and the deduced amino acid sequence are 82% homologous to those of human vWF. Cysteine residues #471, 474, 509 and 695, which form intrachain bonds in human vWF, are also present in the bovine vWF sequence.

Calf augmentation and correction of contour deformities.

In general, augmentation of soft tissue has been a problem for plastic surgeons because of the body's protective mechanisms against foreign material, but silicone rubber implants are the author's choice of the materials currently available. The aesthetic considerations of the shape of the calf and techniques and complications are discussed. This article is generously illustrated with case examples.

Cyclic biaxial strain of pulmonary artery endothelial cells causes an increase in cell layer-associated fibronectin.

Bovine pulmonary artery endothelial (PAE) cells were cultured on an artificial compliant substrate (Mitrathane) and were strained biaxially at a frequency of 1/s for 2, 4, 6, 7, or 24 h. Total protein synthesis, determined by estimating the incorporation of radiolabeled precursors into nondialyzable protein, was increased in cultures that had been biaxially strained for 6, 7, or 24 h, with differences more apparent in the cell layer fraction than in the medium fraction. Medium and cell layer-associated fibronectin were quantitated by enzyme-linked immunosorbent assay and by densitometric analysis of the autoradiograms of electrophoresed protein. Fibronectin levels in the medium of biaxially strained cells were initially depressed in comparison to nonstrained controls but, with time, began to approach control values. Cell layer-associated fibronectin of biaxially strained cultures was significantly elevated at 24 h, whereas DNA synthesis was not altered. Immunohistochemical localization of fibronectin and factor VIII-von Willebrand antigen revealed a more intense staining pattern in strained cultures. Distribution of stress fibers containing fibrous actin was visualized by staining with rhodamine-phalloidin and was altered in strained cultures. These observations indicate that cells respond to cyclic biaxial strain by selectively enhancing structural components associated with cell adhesion.

Production and characterization of a monoclonal antibody to human type III collagen.

Human type III collagen from placenta was isolated and purified for use as an immunogen. A monoclonal antibody was produced which specifically recognizes epitopes unique to type III collagen. The specificity of the antibody was determined by inhibition ELISA, an immunoblot assay, and by immunoprecipitation. Results indicated that the monoclonal antibody recognized only the alpha 1(III) polypeptide chains and did not crossreact with type I, IV, or V collagen. The monoclonal antibody was also used for immunohistochemical localization of type III collagen in tissue sections of human placenta, bovine spleen, and lymph node. In placenta, both large and small blood vessels showed pronounced staining of the tunica media, which contains largely smooth muscle cells, known to synthesize type III collagen. In contrast, the intimal areas and endothelial cells showed no staining with the antibody. In the placental villi, staining was limited to the villous core, where fine fibrillar structures showed strong staining. In lymph nodes, the capsule and pericapsular adipose cells were surrounded by a covering of type III collagen. Within the parenchyma of the node, staining was localized to a branching, reticular array of fine fibers. In the spleen, staining was pronounced in the capsule, splenic trabeculae, and white pulp, where blood vessel staining was especially prominent. The red pulp and splenic sinuses contain little or no type III collagen. The fine network-like or reticular staining pattern found in the lymph node parenchyma is consistent with the staining pattern of the protein reticulin, and suggests that type III collagen may be closely associated with reticulin in certain tissues. Since the role of type III in tissues is unclear, this reagent will be useful in providing new information in this regard.

Immunochemical characterization of type IV procollagen from anterior lens capsule.

Basement membrane collagen from bovine anterior lens capsules (ALC) was isolated by a non-degradative procedure and characterized. The material was obtained by extracting the ALC with 0.5 M acetic acid, passing the extract through a DEAE-cellulose column, collecting and concentrating the unbound fraction. Amino acid and carbohydrate analysis, polyacrylamide gel electrophoresis and rotary shadowing electron microscopy showed the purified extract to have the characteristics of basement membrane procollagen. Examination by rotary shadowing revealed the material to consist of type IV procollagen-like tetramers in various degrees of aggregation. When this purified procollagen was injected into rabbits, antibody of high titer and specificity was obtained. The competitive ELISA was then used to demonstrate lack of reactivity of the antibody with collagen types I, II, III and V and fibronectin. The electroimmunoblot technique was used to demonstrate lack of reactivity with laminin. Competition with collagenase resistant fragments of type IV procollagen representing the carboxyl terminal domain (NCI) and the 7-S domain, as well as with a pepsin-resistant alpha 1 (IV)125K chain was markedly weaker as compared to the native type IV procollagen molecule.