Use of Kirschner wire for immediate interim reconstruction of continuity defect of mandible in resource poor setting: Our experience.

Background: Reconstruction of continuity defect of mandible is challenging, and there has been a lot of advancement in this field with variety of options for restoration. However, choice often becomes restricted in resource limited settings due to lack of trained manpower, time, infrastructure, supply of graft materials, etc. In this context, we aim to test the reliability of Kirschner wire (K-wire) with adaptation of looped-end for immediate interim reconstruction of continuity defect of mandible. Methods: Retrospectively, medical records of 10 years data were reviewed to include 22 patients who underwent immediate interim reconstruction of mandible with K-wire with looped-end adaptation for continuity defect of mandible. Data regarding patient demographic, type and length of mandibular defect, diameter of the wire and complications were recorded. Result: Among 22 patients within a follow-up period of 1-60 months (median 7.5), complications developed in 7 (31.8%) patients. Three patients (13.6%) had major complications which required interventions, and 5 patients (22.3%) with minor complications were managed conservatively. Total wire removal in our study is only 9%, which is lowest among the reported literature, migration or extrusion through bone was nil, and overall aesthetic and functional result was good in majority except few where either wire had to be removed or got deformed. Conclusion: Kirschner wire (K-wire) should be considered in resource limited setting as an immediate interim reconstructive method of mandible for being cheap and widely available. Our technique of looped-end adaptation results in better stabilisation leading to less removal rate, migration or extrusion.

Reliability of the pectoralis major myocutaneous flap in reconstructive oral cancer surgery in developing countries: Our experience.

BACKGROUND: Although free flaps are nowadays considered 'Gold standard' of head and neck reconstruction, pectoralis major myocutaneous (PMMC) flap is still popular among many reconstructive cancer surgeons in developing countries for its many advantages and also due to lack of resources for free flaps in most of the centers, large number of cancer patients with poor nutritional status and economic condition. However, many studies have reported high complication rate in PMMC flap. So, the purpose of our study was to evaluate the reliability of PMMC flap. METHODS: Within a span of 2 years, 20 reconstructions were done with PMMC flaps in patients with oral cancer and they were followed for a period of 1 year. Documentation was done for patient demographics, site of lesion, duration for reconstruction, occurrence of complications, etc. RESULT: Among 17 males and 3 female patients, complications developed in 4 males and all female patients (total 7 patients, overall 35%). Flap-related complications were - one major (5%) and six minor (30%), which were comprised of three orocutaneous fistula (15%), three partial flap loss (15%), two marginal necrosis (10%), and one donor site necrosis (5%). Total necrosis was nil in our study. All the complications were managed conservatively except the patient with major complication which required intervention. Final cosmetic and functional outcome was acceptable in majority of patients. CONCLUSION: PMMC flap is still 'workhorse' of reconstruction in head neck cancer patients in developing countries and can be used effectively with acceptable morbidity.

Intranuclear matrix metalloproteinases promote DNA damage and apoptosis induced by oxygen-glucose deprivation in neurons.

Degradation of the extracellular matrix by elevated matrix metalloproteinase (MMP) activity following ischemia/reperfusion is implicated in blood-brain barrier disruption and neuronal death. In contrast to their characterized extracellular roles, we previously reported that elevated intranuclear MMP-2 and -9 (gelatinase) activity degrades nuclear DNA repair proteins and promotes accumulation of oxidative DNA damage in neurons in rat brain at 3-h reperfusion after ischemic stroke. Here, we report that treatment with a broad-spectrum MMP inhibitor significantly reduced neuronal apoptosis in rat ischemic hemispheres at 48-h reperfusion after a 90-min middle cerebral artery occlusion (MCAO). Since extracellular gelatinases in brain tissue are known to be neurotoxic during acute stroke, the contribution of intranuclear MMP-2 and -9 activities in neurons to neuronal apoptosis has been unclear. To confirm and extend our in vivo observations, oxygen-glucose deprivation (OGD), an in vitro model of ischemia/reperfusion, was employed. Primary cortical neurons were subjected to 2-h OGD with reoxygenation. Increased intranuclear gelatinase activity was detected immediately after reoxygenation onset and was maximal at 24h, while extracellular gelatinase levels remained unchanged. We detected elevated levels of both MMP-2 and -9 in neuronal nuclear extracts and gelatinase activity in neurons co-localized primarily with MMP-2. We found a marked decrease in PARP1, XRCC1, and OGG1, and decreased PARP1 activity. Pretreatment of neurons with selective MMP-2/9 inhibitor II significantly decreased gelatinase activity and downregulation of DNA repair enzymes, decreased accumulation of oxidative DNA damage, and promoted neuronal survival after OGD. Our results confirm the nuclear localization of gelatinases and their nuclear substrates observed in an animal stroke model, further supporting a novel role for intranuclear gelatinase activity in an intrinsic apoptotic pathway in neurons during acute stroke injury.

Altered claudin expression is a feature of chronic plaque psoriasis.

Epithelial tight junctions play a central role in cell-cell adhesion and are necessary for the selective paracellular movement of ions. Claudins are key components of tight junctions and their expression is altered in gut epithelia in a variety of inflammatory enteropathies, including ulcerative colitis and Crohn's disease. Psoriasis is a chronic inflammatory skin disease affecting approximately 2% of the western population, with significantly increased occurrence in individuals with Crohn's disease. Initial studies investigated the expression of claudins in skin of healthy volunteers and patients with chronic plaque psoriasis. We report here that claudins-1 and -3 are the major protein species present in the epidermis of healthy skin; they are expressed on the surface of epidermal keratinocytes, consistent with their localization to tight junctions. In plaques of psoriasis, claudin-1 was not identifiable in the epidermis, although typical staining patterns were observed in clinically normal, uninvolved skin of patients with psoriasis. Claudin-3 was present in the epidermal granular cell layer in normal skin, but was only identified within the cytosol of epidermal keratinocytes in both involved and uninvolved skin of psoriasis patients. We examined further whether exposure of keratinocytes in vitro to pro-inflammatory cytokines mimicked the observed changes in claudin expression seen in chronic plaque psoriasis; lipopolysaccharide, interferon-gamma and tumour necrosis factor-alpha had no effect on claudin protein expression or distribution. Addition of interleukin-1beta, however, resulted in down-regulation of claudins-1 and -3. Tumour necrosis factor-alpha and interleukin-1beta were further used in an in vivo model of skin inflammation; interleukin-1beta alone modulated claudin protein expression in this system. These data demonstrate that epidermal claudin expression is altered in chronic plaque psoriasis and that expression is in part modulated by interleukin-1beta.

Hermansky-Pudlak syndrome in a pregnant patient.

Hermansky-Pudlak syndrome is a multisystem disorder with albinism, bleeding diathesis and visual impairment as the main features. We report a case of epidural analgesia in a pregnant patient, who was subsequently discovered to have this syndrome. We believe this to be the first such report.

Induction of apoptosis by Phenothiazine derivatives in V79 cells.

Phenothiazine derivatives chlorpromazine (cpz) and trifluoperazine (tfp) were found to induce apoptosis, abnormal cell cycle and expression of p53 in Chinese hamster lung fibroblast V79 cells. Both the drugs can induce apoptosis when cells are treated with drug at a concentration of 10 microg/ml within 4 h, as detected by propidium iodide staining and DNA fragmentation analysis. Flow cytometric analysis revealed that the apoptotic response is mediated by a loss of G(1) population of cells. In Western blot analysis, p21 is induced and p53 is accompanied by additional bands. Also indirect immunolabeling of single cells revealed that p21 is accumulated from cytoplasm into nucleus after the drug treatment and the intensities of p53 increased. Our findings demonstrate for the first time that phenothiazine derivatives, in addition to their cytotoxic effects, could induce apoptosis, an observation that has important clinical implications.

Functional analysis of B144/LST1: a gene in the tumor necrosis factor cluster that induces formation of long filopodia in eukaryotic cells.

B144/LST1 is a gene encoded in the human major histocompatibility complex that produces multiple forms of alternatively spliced mRNA and encodes peptides fewer than 100 amino acids in length. B144/LST1 is strongly expressed in dendritic cells. Transfection of B144/LST1 into a variety of cells induces morphologic changes including the production of long, thin filopodia differing from those seen on transfection of a dominant active CDC42 gene. The structures are dynamically rearranging and sometimes connect one cell with another. The full effect of B144/LST1 protein on cell morphology requires the retention of at least one of the four cysteines of the peptide plus the presence of a hydrophobic segment in the protein, but requires only one of the two coding regions present in the terminal 3' exons.

Homocysteine induces expression and secretion of monocyte chemoattractant protein-1 and interleukin-8 in human aortic endothelial cells: implications for vascular disease.

BACKGROUND: Proinflammatory cytokines play key roles in atherogenesis and disease progression. Because hyperhomocysteinemia is an independent risk factor for cardiovascular disease, we hypothesized that homocysteine could be atherogenic by altering the expression of specific cytokines in vascular endothelial cells. METHODS AND RESULTS: Northern blot and RNase protection assays showed that DL-homocysteine induced mRNA expression of the proinflammatory cytokines monocyte chemoattractant protein-1 (MCP-1) and interleukin-8 (IL-8) in cultured human aortic endothelial cells (HAECs). Homocysteine had no effect on expression of other cytokines, namely tumor necrosis factor-alpha, granulocyte-macrophage colony-stimulating factor, interleukin-1beta, and transforming growth factor-beta. MCP-1 mRNA expression increased 1 hour after homocysteine treatment, reached a maximum within 2 to 4 hours, and declined to basal levels over the next 24 hours. Induction of mRNA expression for both chemokines was observed with as little as 10 micromol/L DL-homocysteine, and maximal expression was achieved with 50 micromol/L DL-homocysteine. Homocysteine also triggered the release of MCP-1 and IL-8 protein from HAECs into the culture medium. The induction was specific for homocysteine, because equimolar concentrations of L-homocystine, L-cysteine, and L-methionine had no effect on mRNA levels and protein release. Furthermore, L-homocysteine induced chemokine expression, but D-homocysteine did not, thus demonstrating enantiomeric specificity. The culture medium from homocysteine-treated HAECs promoted chemotaxis in human peripheral blood monocytes and U937 cells. Anti-human recombinant MCP-1 antibody blocked the migration. CONCLUSIONS: Pathophysiological levels of L-homocysteine alter endothelial cell function by upregulating MCP-1 and IL-8 expression and secretion. This suggests that L-homocysteine may contribute to the initiation and progression of vascular disease by promoting leukocyte recruitment.

Homocysteine metabolism in cardiovascular cells and tissues: implications for hyperhomocysteinemia and cardiovascular disease.

We have determined the activity and protein levels of CBS in a number of cardiovascular cells and tissues by direct enzyme assay and Western blot analysis, respectively. We have also determined the activity of BHMT in these same tissues and cells and have come to the conclusion that neither enzyme is expressed. This results suggests that in the human cardiovascular system homocysteine metabolism is limited to the remethylation pathway catalyzed by MS. Thus, hyperhomocysteinemia in conjunction with a limited metabolic capacity for homocysteine in the cardiovascular system could result in cellular dysfunction.

Structural organisations of hemoglobin and myoglobin influence their binding behaviour with phenothiazines.

Binding modalities of chlorpromazine and trifluoperazine, two widely used antipsychotic phenothiazine drugs with hemoglobin and myoglobin have been studied to understand how the quaternary, tertiary and secondary structural organisations of the proteins regulate the binding process. NaCl-induced alteration in the quaternary structure of hemoglobin influences its binding modality with phenothiazines. Minor alterations in the tertiary structure of thermally denatured myoglobin (denaturation temperature ranging between 30-70 degrees C) do not affect its affinity and the modality of binding with the drugs, but alterations in the secondary structure of the protein denatured at temperatures between 70-80 degrees C influence its binding.

Over expression of inducible proteins in Escherichia coli by treatment with ethanol.

It is reported that ethanol enhances DNA synthesis in E. coli cells [Basu, T and Poddar, R. K. (1994), Folia. Microbiol. 39, 3-6]. This communication reports that during growth of E. coli in the presence of 5% v/v ethanol, the derepressed expression of the cytoplasmic enzymes beta-galactosidase and D-serine deaminase per cell increased approximately three fold, while that of the periplasmic enzyme alkaline phosphatase decreased approximately 40% compared to control cell levels. However, in cells transformed with the plasmid pSM 456, bearing phoA-lacZ fusion, the level of induced synthesis of the hybrid protein PhoA-LacZ, controlled by the phoA promoter, was elevated by 25% in the presence of 5% v/v ethanol. This result suggests that the induction of the alkaline phosphatase precursor has also been enhanced by the ethanol treatment, but the inhibition in the export of the precursor across the cytoplasmic membrane, by the influence of ethanol, may represent the reason for the deficient expression of active alkaline phosphatase. It is proposed that there is an ethanol-mediated increase in DNA synthesis, resulting in gene amplification, which may enhance the synthesis of inducible proteins in ethanol-treated cells.

Role of thyroid hormone in the morphological differentiation and maturation of astrocytes: temporal correlation with synthesis and organization of actin.

Morphological changes and the molecular mechanisms associated with the maturation of astrocytes were studied under normal and thyroid hormone-deficient conditions using long-term (30 days) primary cultures derived from the neonatal rat brain. Immunocytochemical staining of cells with a monoclonal antibody specific to glial fibrillary acidic protein demonstrated for the first time that, similar to their maturation in vivo, astrocytes maintained in normal serum-containing medium can undergo complete maturation involving two distinct stages of morphological differentiation (from radial glia to flat polygonal cells with epithelioid morphology and then to mature process-bearing cells with stellate morphology). Deficiency of thyroid hormone delays the first step and totally blocks the second stage of differentiation in the maturation process. Comparative staining of normal and thyroid hormone-deficient astrocytes with filamentous actin-specific fluorescein isothiocyanate-phalloidin and quantitation of the various forms of intracellular actin using an improved DNase I assay demonstrated that maturation of astroglial cells is associated with characteristic alterations in the level of cytoskeletal and noncytoskeletal filamentous (F) actin. In particular, the maintenance of the epithelioid form of the hypothyroid astrocytes is associated with a progressive increase in the level of cytoskeletal F-actin and a concomitant decline in the level of non-cytoskeletal F-actin. Quantitation of actin mRNA by Northern blot analysis and studies on the rate of actin synthesis at various stages of differentiation showed that the initial transformation into the epithelioid form is associated with an increase in the rate of synthesis of actin and the expression of its mRNA, while the final transformation into the nature process-bearing form is correlated with a decline in these parameters. The results indicates that thyroid hormone plays an obligatory role in promoting the differentiation and maturation of astrocytes, and that during this process the hormone regulates the expression of actin and its intracellular organization in a way conducive to morphological differentiation.

Trifluoperazine is more effective than chlorpromazine in releasing oxygen from haemoglobin and myoglobin.

The extent of oxygen release from two heme proteins, haemoglobin and myoglobin have been studied in the presence of trifluoperazine and chlorpromazine (5-1000 microM). At a molar ratio (drug:protein) of 1.5, the release of oxygen from haemoglobin was 4 and 15% in the presence of chlorpromazine and trifluoperazine respectively, while from myoglobin the corresponding values were 20 and 40%. The findings were attributed to the greater extent of local conformational change around tryptophan moieties of each of the proteins induced by trifluoperazine.

Regulation of actin and tubulin gene expression by thyroid hormone during rat brain development.

In the developing brain the active neurite outgrowth during the early phase of synaptogenesis is associated with a thyroid hormone dependent expression of tubulin and actin. In this study, the molecular mechanism of thyroid hormone (TH) action on actin and tubulin gene expression in the developing rat brain has been investigated by comparing the steady state levels of both mRNAs with their respective rates of transcription in cerebra from normal and hypothyroid animals. The developmental profile of actin as well as tubulin mRNAs in both normal and hypothyroid brains display a biphasic pattern, increasing progressively during the first week after birth and declining thereafter. However, hypothyroidism resulted in a significant reduction in the steady state levels of both mRNAs during the first postnatal week. During the second and third weeks, in contrast to their rapid decline in the normal controls, the corresponding decrease in the hypothyroid cerebra was retarded and prolonged resulting in their higher levels under TH-deficient condition. Kinetics of stimulation of actin and tubulin mRNAs in the 5-day hypothyroid cerebra following injection of the optimal dose of TH (200 micrograms T3/100 g body wt.) demonstrated elevation of both mRNAs within 1 h indicating a possible role of TH at the transcriptional level. In vitro transcription experiments by nuclear run off assay unambiguously confirmed that actin gene transcription is depressed in the hypothyroid cerebra compared to normal control. This reduced rate of transcription could be significantly induced in the hypothyroid cerebra by incubation of hypothyroid nuclei with T3 prior to transcription. In contrast, except for a reduced transcription in 5-day hypothyroid nuclei, no effect on tubulin gene transcription was evident at any other age. Moreover preincubation of hypothyroid nuclei from all three ages with T3 had no stimulatory effect on tubulin gene transcription. Analysis of age related changes in the rates of transcription of actin and tubulin genes with their corresponding steady state mRNA levels in normal and hypothyroid developing brain provides strong evidence that although additional modes of regulation may be operative, transcription represents an important level of control for thyroidal regulation of actin gene expression while tubulin gene expression is primarily regulated at post-transcriptional level.

A monoclonal antibody to a cytoskeletal protein selectively recognizing malignant neuroectodermal tumors.

Fusion of myeloma (P3X63-Ag 8.653) cells with spleen cells from BALB/c mice immunized with human neuroblastoma (SK-N-SH) cells yielded a hybridoma clone, referred to as 3XB7, with a unique pattern of reactivity to malignant neuroectodermal tumors except gliomas of low-grade malignancy. Indirect immunofluorescence staining under different conditions and Western blot analysis indicate that the 3XB7 MAb recognizes an intracellular cytoskeletal protein of M(r) 52K. Immunohistochemical studies with cryostat and paraffin-embedded sections from tumor biopsies revealed that the 3XB7 MAb specifically recognizes malignant neuroectodermal tumors and reacts negatively with other epithelial and mesenchymal tumors, e.g., carcinomas, lymphomas, and sarcomas as well as with normal adult and fetal brain tissues. Negative reaction was also observed with other small round cell tumors of childhood. Thus the 3XB7 antigen can be used for diagnosis of all stages of neuroblastomas, and its specific expression in gliomas with high-grade malignancy (grades III and IV) confer on it additional prognostic value.

Interaction of chlorpromazine with myoglobin and hemoglobin. A comparative study.

The mode and nature of the binding of chlorpromazine (CPZ), a psychotropic drug, with myoglobin, a monomeric muscle protein, were studied spectrofluorometrically and the results compared with those from the binding of CPZ to hemoglobin, a tetrameric allosteric protein from red blood cells (RBC). CPZ interacted with myoglobin in a non-cooperative mode, with a binding constant of 8.4 x 10(3) M-1 in 0.145 M NaCl, pH 6.8, whereas in the case of hemoglobin this interaction was found to be positively cooperative with a binding constant of 4.2 x 10(3) M-1. The interaction of CPZ with myoglobin was not influenced by the NaCl molarity of the solution, whereas CPZ interaction with hemoglobin significantly decreased with increasing NaCl molarity, indicating that CPZ-hemoglobin binding is mostly electrostatic in nature, whereas that of the CPZ-myoglobin complex is of a non-electrostatic type. Thermodynamic analysis revealed that binding of CPZ to hemoglobin was exothermic (delta H degrees = -2.65 kcal/mol), whereas binding to myoglobin was endothermic (delta H degrees = + 1.39 kcal/mol) with a high entropic contribution (delta S degrees = +23 cal/degree/mol), suggesting that CPZ binding to myoglobin is hydrophobic in nature. Such contrasting binding features of this drug have been discussed in the light of a typical subunit interaction property present and absent in hemoglobin and myoglobin, respectively.

Cytotoxic and genetic effects of X-irradiation of human cells in the presence of chlorpromazine.

Human epidermoid carcinoma cells (Hep-2) were X-irradiated in the presence of 5-10 micrograms/ml of chlorpromazine (CPZ). Survival of the cells decreased with increasing CPZ concentration. Lymphocytes from three normal volunteers exposed to X-irradiation in the presence of CPZ showed an increased frequency of dicentric and ring formation.