Total lower lip reconstruction with a composite radial forearm-palmaris longus tendon flap: a clinical series

Large, full-thickness lip defects after head and neck surgery continue to be a challenge for reconstructive surgeons. The reconstructive aims are to restore the oral lining, the external cheek, oral competence, and function (i.e., articulation, speech, and mastication). The authors' refinement of the composite radial forearm-palmaris longus free flap technique meets these criteria and allows a functional reconstruction of extensive lip and cheek defects in one stage. A composite radial forearm flap including the palmaris longus tendon was designed. The skin flap for the reconstruction of the intraoral lining and the skin defect was folded over the palmaris longus tendon. Both ends of the vascularized tendon were laid through the bilateral modiolus and anchored with adequate tension to the intact orbicularis muscle of the upper lip. This procedure was used in 12 patients. Six patients had cancer of the lower lip, five patients had a buccal cancer involving the lip, and one patient had a primary gum cancer that extended to the lower lip. Total to near-total resection (more than 80 percent) of the lower lip was indicated in six patients. In two other patients, the cancer ablation included more than 80 percent of the lower lip and up to 40 percent of the upper lip. A radial forearm palmaris longus free flap was used in all cases for reconstruction of the defect. Free flap survival was 100 percent. At the time of final evaluation, which was 1 year after the operation, all patients had good oral continence at rest (static suspension) and had achieved sufficient oral competence when eating. Ten patients were able to resume a regular diet, and two patients could eat a soft diet. All patients regained normal or near-normal speech and had an acceptable appearance. The described refinement of the composite radial palmaris longus free flap technique allows the reconstruction of the lower lip with a functioning oral sphincter; the technique can be recommended for patients who need large lower lip resection. It provides functional recovery of the reconstructed lower lip synchronizing with the remaining upper lip.     

Circular dichroism spectra of human hemoglobin reveal a reversible structural transition at body temperature

Previously we have shown that human red blood cells (RBCs) undergo a sudden change from blocking to passing through a 1.3±0.2-µm micropipette when applying an aspiration pressure of 2.3 kPa at a critical transition temperature (T_c=36.4±0.3 °C). Low-shear viscosity measurements suggested that changes in the molecular properties of hemoglobin might be responsible for this effect. To evaluate structural changes in hemoglobin at the critical temperature, we have used circular dichroism (CD) spectroscopy. The thermal denaturation curves of human hemoglobin A (HbA) and hemoglobin S (HbS) upon heating between 25 and 60 °C were non-linear and showed accelerated denaturation between 35 and 39 °C with a midpoint at 37.2±0.6 °C. The transition was reversible below 39 °C and independent of solution pH (pH 6.8–7.8). It was also independent of the oxygenation state of hemoglobin, since a sample that was extensively deoxygenated with N₂ showed a similar transition by CD. These findings suggest that a structural change in hemoglobin may enable the cellular passage phenomenon as well as the temperature-dependent decrease in viscosity of RBC solutions.     

Small proline-rich protein 1A is a gp130 pathway- and stress-inducible cardioprotective protein

The interleukin-6 cytokines, acting via gp130 receptor pathways, play a pivotal role in the reduction of cardiac injury induced by mechanical stress or ischemia and in promoting subsequent adaptive remodeling of the heart. We have now identified the small proline-rich repeat proteins (SPRR) 1A and 2A as downstream targets of gp130 signaling that are strongly induced in cardiomyocytes responding to biomechanical/ischemic stress. Upregulation of SPRR1A and 2A was markedly reduced in the gp130 cardiomyocyte-restricted knockout mice. In cardiomyocytes, MEK1/2 inhibitors prevented SPRR1A upregulation by gp130 cytokines. Furthermore, binding of NF-IL6 (C/EBPbeta) and c-Jun to the SPRR1A promoter was observed after CT-1 stimulation. Histological analysis revealed that SPRR1A induction after mechanical stress of pressure overload was restricted to myocytes surrounding piecemeal necrotic lesions. A similar expression pattern was found in postinfarcted rat hearts. Both in vitro and in vivo ectopic overexpression of SPRR1A protected cardiomyocytes against ischemic injury. Thus, this study identifies SPRR1A as a novel stress-inducible downstream mediator of gp130 cytokines in cardiomyocytes and documents its cardioprotective effect against ischemic stress.     

Nucleosides. IX. Synthesis of purine N(3),5'-cyclonucleosides and N(3),5'-cyclo-2',3'-seconucleosides via Mitsunobu reaction as TIBO-like derivatives

The Mitsunobu reaction was applied to prepare, in one step, purine N(3),5'-cyclonucleosides 10a-d. A subsequent ring opening in the ribose moiety of the resultant N(3),5'-nucleosides by sodium periodate led to the corresponding N(3),5'-cyclo-2',3'-seconucleosides. These products consist of 5-, 6-, and 7-membered tricyclic system which is the basic skeleton of TIBO derivatives, known antiviral agents.     

Wild type p53 gene causes reorganization of cytoskeleton and,therefore, the impaired deformability and difficult migration of murine erythroleukemia cells

We studied the role of p53 gene in the biophysics and biology in murine erythroleukemia cell line (MEL), with the goal of understanding the influence of this tumor suppressor gene on the deformability and metastasis of tumor cells. Experiments were performed on MEL and p53-transfected MEL (MEL-M with mutant p53 gene and MEL-W with wild-type p53 gene). The cell growth curves indicated that the over-expression of wild-type p53 gene significantly suppressed the growth of MEL, with G(0)-G(1) arrest and apoptosis shown by flow cytometric assays. Confocal laser scanning microscopy revealed that the MEL-W had a more compact organization of the F-Actin cytoskeleton than MEL and MEL-M. Fluorescence polarization measurement indicated a higher membrane fluidity of MEL-W than the other two groups. Fourier transform infrared spectroscopy (FT-IR) showed changes in the composition and/or structure of membrane lipids in MEL-W, with decreases in secondary structures of proteins such as alpha-helix, turns and bends and random coil, in comparison to MEL and MEL-M. The osmotic fragility curves indicated that MEL-W was more fragile and micropipette experiments showed that they had increased elasticity and reduced deformability in comparison to MEL and MEL-M. The adhesion assay with the use of the flow chamber revealed a lower adhesion rate of MEL-W to endothelial cells at high shear stress. The present study on the molecular biology with biophysics of MEL cells contributes to our knowledge on the tumor suppressor gene p53.     

An LKB1-interacting protein negatively regulates TNFα-induced NF-κB activation

The Peutz-Jeghers syndrome (PJS) is a hereditary disorder that predisposes an individual to benign and malignant tumors in multiple organ systems. Recently, the locus responsible for PJS was mapped genetically to the LKB1 gene, with a subsequent investigation proving that it is responsible for most cases of PJS. LKB1 encodes a nuclear serine/threonine protein kinase, and potential tumor-suppressing activity has been attributed to LKB1 kinase. However, how LKB1 exerts its tumor-suppressing function remains to be determined. In this report, we describe the identification of a putative human LKB1-interacting protein, FLIP1, using the yeast two-hybrid system. Two regions of the LKB1 sequence have been determined to be crucial for the interaction with FLIP1. FLIP1 encodes a protein of 429 amino acids with a predicted molecular weight of 47 kd. In contrast to LKB1, which is mainly nuclear, FLIP1 is a cytoplasmic protein, and its expression is ubiquitous in all human tissues examined to date. Interestingly, deletion of the 195 N- terminal amino acids allows FLIP1 to enter the nucleus, suggesting the presence of a regulatory mechanism through its N-terminus for nuclear entry. In addition, we found that ectopic expression of FLIP1 selectively blocks cytokine-induced NF-kappaB activation. The involvement of FLIP1 in the regulation of NF-kappaB activity may shed new light on the role of LKB1 in tumor suppression.     

OxLDL induces mitogen-activated protein kinase activation mediated via PI3-kinase/Akt in vascular smooth muscle cells

Oxidized low-density lipoprotein (OxLDL) is a risk factor in atherosclerosis and stimulates multiple signaling pathways, including activation of phosphatidylinositol 3-kinase (PI3-K)/Akt and p42/p44 mitogen-activated protein kinase (MAPK), which are involved in mitogenesis of vascular smooth muscle cells (VSMCs). We therefore investigated the relationship between PI3-K/Akt and p42/p44 MAPK activation and cell proliferation induced by OxLDL. OxLDL stimulated Akt phosphorylation in a time- and concentration-dependent manner, as determined by Western blot analysis. Phosphorylation of Akt stimulated by OxLDL and epidermal growth factor (EGF) was attenuated by inhibitors of PI3-K (wortmannin and LY294002) and intracellular Ca2+ chelator (BAPTA/AM) plus EDTA. Pretreatment of VSMCs with pertussis toxin, cholera toxin, and forskolin for 24 h also attenuated the OxLDL-stimulated Akt phosphorylation. In addition, pretreatment of VSMCs with wortmannin or LY294002 inhibited OxLDL-stimulated p42/p44 MAPK phosphorylation and [3H]thymidine incorporation. Furthermore, treatment with U0126, an inhibitor of MAPK kinase (MEK)1/2, attenuated the p42/p44 MAPK phosphorylation, but had no effect on Akt activation in response to OxLDL and EGF. Overexpression of p85-DN or Akt-DN mutants attenuated MEK1/2 and p42/p44 MAPK phosphorylation stimulated by OxLDL and EGF. These results suggest that the mitogenic effect of OxLDL is, at least in part, mediated through activation of PI3-K/Akt/MEK/MAPK pathway in VSMCs.     

The role of p53 deacetylation in p21Waf1 regulation by laminar flow

Laminar flow arrests vascular endothelial cells at the G0/G1 phase with concurrent increase in p53 and p21Waf1. We investigated the molecular mechanism by which laminar flow activates p53 and p21Waf1 in endothelial cells. The application of a laminar flow (12 dyn/cm2) increased the deacetylation at Lys-320 and Lys-373 of p53 and the acetylation at Lys-382 in human umbilical vein endothelial cells. Laminar flow increased the activity of histone deacetylase (HDAC) and the association of p53 with HDAC1. Treating human umbilical vein endothelial cells with trichostatin A (TSA), an HDAC inhibitor, abolished the flow-induced p53 deacetylation at Lys-320 and Lys-373. To investigate the role of the HDAC-deacetylated p53 in the flow activation of p21Waf1, we found that TSA inhibited the activation at both the mRNA and protein levels. Deletion and mutation analyses of the p21Waf1 promoter revealed that flow activated p21Waf1 through p53 and TSA abrogated this p53-dependent activation. The expression plasmid encoding the p53 mutant, with Lys-320 and Lys-373 replaced by Arg, increased the activity of the co-transfected p21Waf1 promoter, which demonstrates that HDAC-deacetylated p53 can transactivate the p21Waf1 gene. The regulation of the p53-p21Waf1 pathway by laminar flow was further supported by observations that flow caused an increase of p21Waf1 level in the wild-type HCT116 (p53+/+) cells but not in the p53-null HCT116 cells.     

Atrial chamber-specific expression of sarcolipin is regulated during development and hypertrophic remodeling

Intracellular Ca2+ regulation is critical in the normal cardiac function and development of pathologic hearts. Phospholamban, an endogenous inhibitor of sarcoplasmic reticulum Ca2+ ATPase in the sarcoplasmic reticulum, plays an important role in Ca2+ cycling in heart. Recently, sarcolipin has been identified as having a similar function as phospholamban in skeletal muscle. Because phospholamban is differentially expressed in atrial and ventricular myocardia and its expression is often altered in diseased hearts, we investigated the cardiac chamber specificity of sarcolipin expression and its regulation during development and hypertrophic remodeling. Northern blot analysis revealed that the expression of mouse sarcolipin mRNA was most abundant in the atria and was undetectable in the ventricles, indicating an atrial chamber-specific expression pattern. Atrial chamber-specific expression of sarcolipin mRNA was increased during development. These findings were confirmed by in situ hybridization studies. In addition, sarcolipin expression was down-regulated in the atria of hypertrophic heart when induced by ventricular specific overexpression of the activated H-ras gene. In humans, sarcolipin mRNA was also expressed in the atria but not detected in the ventricles, although sarcolipin expression was most abundant in skeletal muscle. Taken together, sarcolipin is likely to be an atrial chamber-specific regulator of Ca2+ cycling in heart.