Trio Haploinsufficiency Causes Neurodevelopmental Disease-Associated Deficits

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Identification of protease 3.4.24.11 as the major atrial natriuretic factor degrading enzyme in the rat kidney

We have identified a metalloendoprotease from rat kidney cortex that cleaves the cysteine-phenylalanine bond (Cys7-Phe8) within the 17 amino acid ring structure of atrial natriuretic factor (ANF). Cleavage at this site represents the major ANF degradative activity in rat kidney, and is inhibited by the known metalloendoprotease inhibitors, thiorphan, phosphoramidon and zincov with IC50 values in the nanomolar range. Since these are specific inhibitors of protease 3.4.24.11, both protease 3.4.24.11 and ANF degrading activities were monitored during purification. Both activities copurified at each chromatographic step. Furthermore, purified protease 3.4.24.11 cleaved ANF specifically at the Cys7-Phe8 bond. It is concluded from this work that the major ANF degrading enzyme in rat kidney is protease 3.4.24.11.     

Effects of nitric oxide synthase inhibitors on systemic hypotension, cytokines and inducible nitric oxide synthase expression and lung injury following endotoxin administration in rats

Endotoxin shock is characterized by systemic hypotension, hyporeactiveness to vasoconstrictors and acute lung edema. A nitric oxide synthase (NOS) inhibitor, N^G-monomethyl-L-arginine (L-NMMA) has been shown to be effective in reversing acute lung injury. In the present study, we evaluated the effects of NOS blockade by different mechanisms on the endotoxin-induced changes. In anesthetized rats, lipopolysaccharide (LPS,Klebsiella pneumoniae) was administered intravenously in a dose of 10 mg/kg. LPS caused sustained systemic hypotension accompanied by an eightfold increase of exhaled NO during an observation period of 4 h. After the experiment, the lung weight was obtained and lung tissues were taken for the determination of mRNA expressions of inducible NOS (iNOS), interleukin-1 (IL-1) and tumor necrosis factor--(TNF-). Histological examination of the lungs was also performed. In the control group injected with saline solution, mRNA expressions of iNOS, IL-1 and TNF- were absent. Four hours after LPS, the mRNA expressions of iNOS and IL-1 were still significantly enhanced, but TNF- was not discernibly expressed. LPS also caused a twofold increase in lung weight. Pathological examination revealed endothelial damage and interstitial edema. Various NOS inhibitors were given 1 h after LPS administration. These agents included N-nitro-L-arginine methyl ester (L-NAME, 10 mg/kg), a constitutive NOS and iNOS inhibitor; S,S-1,4-phenylene-bis-(1,2-ethanedinyl) bis-isothiourea dihydrobromide (1,4-PBIT, 10 mg/kg), a relatively specific iNOS inhibitor, and dexamethasone (3 mg/kg), an inhibitor of iNOS expression. These NOS inhibitors all effectively reversed the systemic hypotension, reduced the exhaled NO concentration and prevented acute lung injury. The LPS-induced mRNA expressions of iNOS and IL-1 were also significantly depressed by these NOS inhibitors. Our results suggest that NO production through the iNOS pathway is responsible for endotoxin-induced lung injury. Certain cytokines such as IL-1 are possibly involved. These changes are minimized by NOS inhibitors through different mechanisms.     

Protease-activated receptor-1-induced calcium signaling in gingival fibroblasts is mediated by sarcoplasmic reticulum calcium release and extracellular calcium influx

Thrombin is a serine protease activated during injury and inflammation. Thrombin and other proteases generated by periodontal pathogens affect the behavior of periodontal cells via activation of protease-activated receptors (PARs). We noted that thrombin and PAR-1 agonist peptide stimulated intracellular calcium levels ([Ca2+]i) of gingival fibroblasts (GF). This increase of [Ca2+]i was inhibited by EGTA and verapamil. U73122 and neomycin inhibited thrombin- and PAR-1-induced [Ca2+]i. Furthermore, 2-APB (75-100 microM, inositol triphosphate [IP3] receptor antagonist), thapsigargin (1 microM), SKF-96365 (200 microM) and W7 (50 and 100 microM) also suppressed the PAR-1- and thrombin-induced [Ca2+]i. However, H7 (100, 200 microM) and ryanodine showed little effects. Blocking Ca2+ efflux from mitochondria by CGP37157 (50, 100 microM) inhibited both thrombin- and PAR-1-induced [Ca2+]i. Thrombin induced the IP3 production of GF within 30-seconds of exposure, which was inhibited by U73122. These results indicate that mitochondrial calcium efflux and calcium-calmodulin pathways are related to thrombin and PAR-1 induced [Ca2+]i in GF. Thrombin-induced [Ca2+]i of GF is mainly due to PAR-1 activation, extracellular calcium influx via L-type calcium channel, PLC activation, then IP3 binding to IP3 receptor in sarcoplasmic reticulum, which leads to intracellular calcium release and subsequently alters cell membrane capacitative calcium entry.     

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.     

Fingertip replantation using the subdermal pocket procedure

Restoration of finger length and function are the goals of replantation after fingertip amputation. Methods include microsurgical replantation and nonmicrosurgical replantation, such as composite graft techniques. To increase the survival rates for composite grafts, the subcutaneous pocket procedure has been used as a salvage procedure. The subdermal pocket procedure, which is a modification of the subcutaneous pocket procedure, was used for replantation of 17 fingertips in 16 consecutive patients. Eight fingertips experienced guillotine injuries and the other nine fingertips experienced crush injuries. Revascularization of one digital artery without available venous outflow was performed for six fingers, and composite graft techniques were used for the other 11 fingers. The success rate was 16 of 17 cases. The difference in success rates for guillotine versus crush injuries was statistically significant. Comparison of patients with arterial anastomoses and patients without arterial anastomoses also indicated a statistically significant difference. Thirteen fingertips survived completely. One finger, demonstrating complete loss and early termination of the pocketing procedure, was amputated on the eighth postoperative day. Two fingers were partially lost because of severe crushing injuries. One finger demonstrated partial loss of more than one quarter of the fingertip, which required secondary revision, because the patient was a heavy smoker. The pocketing period was 8 +/- 1 days (mean +/- SD, n = 6) for the fingers revascularized with one digital arterial anastomosis and 13.3 +/- 1.9 days (n = 10) for the fingers successfully replanted with composite graft techniques. The mean active range of motion of the interphalangeal joint of the three thumbs was 65 +/- 5 degrees, and that of the distal interphalangeal joint of the other 11 fingers was 51 +/- 11 degrees. The static two-point discrimination result was 6.4 +/- 1.0 mm (n = 14) after an average of 11 +/- 5 months of follow-up monitoring. Compared with other methods, the subdermal pocket procedure has the advantages of exact subdermal/subdermal contact, a shorter pocketing period, and more feasible observation. The method can offer an alternative salvage procedure for fingertip amputations with no suitable vessels available for microsurgical replantation.     

DNA-induced aggregation of zwitterionic oligolamellar liposome

Liposome consisting of a single zwitterionic lipid as the potential vector for gene therapy has been reported recently; however, whether polyanionic DNA can bind directly with zwitterionic lipid without the aid of multivalent salt still remains unresolved. In this study, we reveal the aggregation of zwitterionic oligolamellar liposomes composed of 1,2-di(cis-9-octadecenoyl)-sn-glycero-3-phosphocholine induced by DNA without the presence of multivalent salt. Our results demonstrate that only a small fraction (<10%) of DNA can bind electrostatically with a portion of the liposomes. Such a low degree of binding, however, induces significant aggregation of these oligolamellar liposomes, yielding large multilamellar particles in which the number of hydrophilic/hydrophobic layer stacking becomes sufficiently large to yield multiple diffraction peaks in the small-angle X-ray scattering profile. Addition of monovalent salt such as NaCl tends to disrupt the multilamellar structure.     

Free anterolateral thigh flap for extremity reconstruction: clinical experience and functional assessment of donor site

From August of 1995 through July of 1998, 38 free anterolateral thigh flaps were transferred to reconstruct soft-tissue defects. The overall success rate was 97 percent. Among 38 anterolateral thigh flaps, four were elevated as cutaneous flaps based on the septocutaneous perforators. The other 34 were harvested as myocutaneous flaps including a cuff of vastus lateralis muscle (15 to 40 cm3), either because of bulk requirements (33 cases) or because of the absence of a septocutaneous perforator (one case). However, vastus lateralis muscle is the largest compartment of the quadriceps, which is the prime extensor of the knee. Losing a portion of the vastus lateralis muscle may affect knee stability. Objective functional assessments of the donor sites were performed at least 6 months postoperatively in 20 patients who had a cuff of vastus lateralis muscle incorporated as part of the myocutaneous flap; assessments were made using a kinetic communicator machine. The isometric power test of the ratios of quadriceps muscle at 30 and 60 degrees of flexion between donor and normal thighs revealed no significant difference (p > 0.05). The isokinetic peak torque ratio of the quadriceps and hamstring muscles, including concentric and eccentric contraction tests, showed no significant difference (p > 0.05), except the concentric contraction test of the quadriceps muscle, which revealed mild weakness of the donor thigh (p < 0.05). In summary, the functional impairment of the donor thighs was minimal after free anterolateral thigh myocutaneous flap transfer.     

Reactive thrombocytosis alone does not affect the patency of microvascular anastomosis in the splenectomy rat

Vascular thrombosis is a harbinger of failure in microsurgery. However, there is still controversy regarding the correlation of the complications of thrombocytosis and thrombosis. Some evidence indicates that patients with elevated platelet counts tend to have a higher flap failure rate, and surgeons usually hesitate to operate on patients with thrombocytosis. Nevertheless, the authors have experienced successful free tissue transfer in seven patients with thrombocytosis resulting from traumatic splenectomy or multiple trauma. On the basis of clinical observation, the authors investigated whether reactive thrombocytosis contributes to the patency of a microvascular anastomosis. In a rodent splenectomy-induced thrombocytosis model (n = 40), stable reactive thrombocytosis occurred after postoperative days 5 to 10, with the peak on postoperative day 7. Femoral artery division and reanastomosis was performed in rats with or without splenectomy-induced thrombocytosis, and vascular patency was assessed. Platelet counts and platelet activation were studied in correlation to microvascular patency. Platelet activation as demonstrated by CD62P expression on platelets was not significantly different between rats with and without thrombocytosis (6.41 +/- 0.95 percent versus 4.51 +/- 0.55 percent, respectively; p = 0.089). As immature platelets were not increased (2.86 +/- 0.33 percent versus 1.99 +/- 0.32 percent, p = 0.074), it seems that the splenectomy-induced thrombocytosis is the result of redistribution of platelets instead of an increase in bone marrow production. There were no significant differences in the patency rates or perfusion units of femoral artery after arterial anastomosis between rats with and without thrombocytosis (90 percent and 95 percent, respectively; p = 0.561). In conclusion, this study demonstrates that microvascular anastomosis can be performed safely in patients with reactive thrombocytosis without platelet activation.