[Clinical characteristics and analysis of risk factors for heart injuries in 55 patients with lightning injury on plateau in Tibet Autonomous Region].

Objective: To explore the clinical characteristics and treatment outcomes of patients with lightning injury on plateau in Tibet Autonomous Region, and to analyze the risk factors for heart injuries in these patients. Methods: A retrospective case series study was conducted. From January 2008 to July 2023, 55 patients with lightning injury who met the inclusion criteria were admitted to the General Hospital of PLA Tibet Military Area Command. The gender, age, ethnicity, time of injury, location of injury (average altitude), activity at the time of injury, the occurrence of thermal burns on the body surface, the occurrence of complication, the occurrence of combined injury, underlying disease or physiological process before injury, length of hospital stay, treatment outcome, and effective rate of treatment were recorded. The patients were divided into juvenile group (11 cases), young group (28 cases), middle-aged group (14 cases), and elderly group (2 cases) according to age bracket, then the gender and ethnicity distribution of patients in the 4 groups were compared. According to the occurrence of heart injuries at admission, the patients were divided into heart injury group (44 cases) and non-heart injury group (11 cases), then the gender, age, ethnicity, average altitude of location of injury, length of hospital stay, the occurrence of complication, the occurrence of combined injury, site of thermal burns on the body surface, and area of thermal burns on the body surface in patients were compared between the two groups. Data were statistically analyzed with Mann-Whitney U test, chi-square test, or Fisher's exact probability test. The multivariate logistic regression analysis was conducted to screen the independent risk factors for heart injury in patients with lightning injury. Results: Among the 55 patients aged 10-68 years, 39 were male and 16 were female, including 47 Tibetans and 8 Hans. There were no statistically significant differences in gender or ethnicity distribution of patients among the 4 groups with different age brackets (P>0.05). Lightning injuries occurred from May to September, which mostly occurred in June and July. The incidence of lightning injury was higher in Chengguan District of Lhasa City (average altitude of 3 650 m) and Baqing County of Naqu City (average altitude of 4 500 m), being 20.0% (11/55) and 16.4% (9/55), respectively. A total of 96.4% (53/55) of the patients were engaged in outdoor activities when injured, such as grazing, digging Cordyceps, and harvesting highland barley. Among the 55 patients, 46 (83.6%) cases had thermal burns on the body surface, with burn area mainly being not more than 10% total body surface area and burn depth mainly being deep partial-thickness. Fifty-two (94.5%) patients had complications, with heart injury being the most common complication (44 cases, 80.0%). Twenty-two (40.0%) patients had 11 combined injuries, and traumatic brain injury was the most common combined injury. Seventeen (30.9%) patients had 11 underlying diseases or physiological processes before injury. The length of hospital stay of patients was 9 (5, 17) d. Among the 55 patients, 14 cases were cured and discharged, 40 cases were improved, and 1 case died, with effective rate of treatment of 98.2%. Compared with those in non-heart injury group, the proportion of complication occurrence (χ2=12.28), the proportion of trunk burns (χ2=5.15), and the average altitude of location of injury (Z=-2.38) of patients in heart injury group were increased significantly (P<0.05), while there were no significant changes in the other indicators (P>0.05). Multivariate logistic regression analysis showed that the average altitude at the location of injury was the independent risk factor for heart injury in patients with lightning injury (with odds ratio of 3.28, 95% confidence interval of 1.35-7.99, P<0.05). Conclusions: Lightning injuries on plateau in Tibet Autonomous Region mainly occur from May to September, with an average altitude of 4 500 m at the location of injury. Patients with lightning injury are injured when participating outdoor activities, and the affected patients are mainly mainly young male Tibetans. Most of the injuries are mild burns. Lightning injuries are complex and have many complications, with heart injury being the most common one. The average altitude at the location of injury is the independent risk factor for heart injury in patients with lightning injury.

The Convective Transport of Active Species in the Tropics (CONTRAST) Experiment.

The Convective Transport of Active Species in the Tropics (CONTRAST) experiment was conducted from Guam (13.5° N, 144.8° E) during January-February 2014. Using the NSF/NCAR Gulfstream V research aircraft, the experiment investigated the photochemical environment over the tropical western Pacific (TWP) warm pool, a region of massive deep convection and the major pathway for air to enter the stratosphere during Northern Hemisphere (NH) winter. The new observations provide a wealth of information for quantifying the influence of convection on the vertical distributions of active species. The airborne in situ measurements up to 15 km altitude fill a significant gap by characterizing the abundance and altitude variation of a wide suite of trace gases. These measurements, together with observations of dynamical and microphysical parameters, provide significant new data for constraining and evaluating global chemistry climate models. Measurements include precursor and product gas species of reactive halogen compounds that impact ozone in the upper troposphere/lower stratosphere. High accuracy, in-situ measurements of ozone obtained during CONTRAST quantify ozone concentration profiles in the UT, where previous observations from balloon-borne ozonesondes were often near or below the limit of detection. CONTRAST was one of the three coordinated experiments to observe the TWP during January-February 2014. Together, CONTRAST, ATTREX and CAST, using complementary capabilities of the three aircraft platforms as well as ground-based instrumentation, provide a comprehensive quantification of the regional distribution and vertical structure of natural and pollutant trace gases in the TWP during NH winter, from the oceanic boundary to the lower stratosphere.

Eicosapentaenoic acid attenuates dexamethasome-induced apoptosis by inducing adaptive autophagy via GPR120 in murine bone marrow-derived mesenchymal stem cells.

Long-term use of glucocorticoids is a widespread clinical problem, which currently has no effective solution other than discontinuing the use. Eicosapentaenoic acid (EPA), an omega-3 long chain polyunsaturated fatty acid (n-3 PUFA), which is largely contained in fish or fish oil, has been reported to promote cell viability and improve bone metabolism. However, little is known about the effects of EPA on dexamethasome (Dex)-induced cell apoptosis. In this study, we showed that EPA-induced autophagy of murine bone marrow-derived mesenchymal stem cells (mBMMSCs). Meanwhile, EPA, but not arachidonic acid (AA), markedly inhibited Dex-induced apoptosis and promoted the viability of mBMMSCs. We also observed that EPA-induced autophagy was modulated by GPR120, but not GPR40. Further experiments showed that the mechanism of EPA-induced autophagy associated with GPR120 modulation involved an increase in the active form of AMP-activated protein kinase and a decrease in the activity of mammalian target of RAPA. The protective effect of EPA on Dex-induced apoptosis via GPR120-meditated induction of adaptive autophagy was supported by in vivo experiments. In summary, our findings may have important implications in developing future strategies to use EPA in the prevention and therapy of the side effects induced by long-term Dex-abuse.

EGb761 improves histological and functional recovery in rats with acute spinal cord contusion injury.

STUDY DESIGN: This is an experimental study. OBJECTIVES: The objective of this study was to evaluate the neuroprotective effects of Ginkgo biloba extract 761 (EGb761) on histological features of injured sites and on functional performance of rats subjected to standardized spinal cord injury (SCI). SETTING: This study was conducted in Xian, Shaanxi, China. METHODS: Thirty female Sprague-Dawley rats were randomly divided into three groups: sham-operated, saline-treated control and EGb761-treated. The Basso, Beattie, Bresnahan Locomotor Rating Score (BBB score) was calculated and footprint analysis was performed to evaluate the functional performance of the rats in each group. Hematoxylin and eosin (HE) staining and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and caspase-3 staining were performed to evaluate the necrosis area and apoptotic cells at the injured site in each group. RESULTS: At 14, but not 1, 3 and 7, days post injury (DPI), rats in the EGb761-treated group exhibited significantly better BBB scores compared with the saline-treated control group (P<0.05). The EGb761-treated group also showed increased stride length, decreased stride width and reduced toe dragging at 14 DPI (P<0.05). Analysis of HE staining revealed that the EGb761-treated group had reduced necrosis at the injury site compared with the saline-treated control group (P<0.05). Analysis of TUNEL and caspase-3 staining demonstrated that cell apoptosis was increased at 1-14 DPI, peaking at 24-h post injury in the gray matter, and 7 DPI in the white matter. At 7 DPI, the quantity of apoptotic cells was significantly decreased in the EGb761-treated group. CONCLUSION: EGb761 administration during the acute phase after SCI significantly reduced secondary injury-induced tissue necrosis and cell apoptosis and improved functional performance in rats.

Associations of depression with impaired glucose regulation, newly diagnosed diabetes and previously diagnosed diabetes in Chinese adults.

AIM: To examine the association between depression and impaired glucose regulation, newly diagnosed diabetes and previously diagnosed diabetes in middle-aged and elderly Chinese people, and whether depression was associated with different treatment regimens or durations of diabetes. METHODS: A cross-sectional study was performed among 229,047 adults living in the community aged ≥ 40 years from 25 centres in China. The self-reported depression rating scale Patient Health Questionnaire 9 (PHQ-9) was used to diagnose probable and sub-threshold depression. Glucose metabolism status was determined according to World Health Organization 1999 diagnostic criteria. RESULTS: The numbers of participants with normal glucose regulation, impaired glucose regulation, newly diagnosed diabetes and previously diagnosed diabetes were 120,458, 59,512, 24,826 and 24,251, respectively. The prevalence of sub-threshold depression in the total sample of participants was 4.8% (4.8%, 4.8%, 4.4% and 5.6% from normal glucose regulation to previously diagnosed diabetes, respectively), and the prevalence of probable depression was 1.1% (1.1%, 1.0%, 0.9% and 1.8% from normal glucose regulation to previously diagnosed diabetes, respectively). Compared with participants with normal glucose regulation, those with previously diagnosed diabetes had increased odds of probable depression [odds ratio (OR) = 1.61, 95% confidence interval (CI) 1.39-1.87] and sub-threshold depression (OR = 1.14, 95% CI 1.06-1.24), after adjustment for multiple confounding factors. Newly diagnosed diabetes or impaired glucose regulation was not associated with depression. Among those with previously diagnosed diabetes, insulin treatment was associated with greater odds of depression compared with no treatment or oral anti-diabetic medicine. CONCLUSION: Previously diagnosed diabetes, but not newly diagnosed diabetes or impaired glucose regulation, was associated with a higher prevalence of depression. Patients receiving insulin were more likely to have depression than those not receiving treatment or being treated with oral anti-diabetic medicine.

High-dose ascorbic acid administration improves functional recovery in rats with spinal cord contusion injury.

OBJECTIVES: To evaluate the effects of different doses of ascorbic acid (AA) on the functional performance of rats subjected to standardized spinal cord injury (SCI). METHODS: Thirty female Sprague-Dawley rats were divided into three groups (10 animals in each group): control group: rats were subjected to SCI injury and received intraperitoneal saline administration; normal-dose AA group: rats were subjected to SCI injury and received daily intraperitoneal administration of AA at 100 mg kg(-1) bodyweight; high-dose AA group: rats were subjected to SCI injury and received daily intraperitoneal administration of AA at 200 mg kg(-1) bodyweight. The Basso, Beattie, Bresnahan Locomotor Rating Score (BBB score) and footprint analysis were performed to evaluate the functional performance of the rats in each group, and hematoxylin and eosin staining was performed to evaluate necrosis at the injury site. RESULTS: At days 14 and 28 after SCI, rats in the high-dose AA group, but not the normal-dose AA group, exhibited significantly better BBB score compared with the control group (P<0.05). Compared with the control and normal-dose AA group, the high-dose AA group also showed increased stride length, decreased stride width and reduced toe dragging (P<0.05). Histological analysis revealed that both the normal- and high-dose AA groups had reduced necrosis in the injury site compared with the control group (P<0.05). CONCLUSION: High-dose AA administration during the acute phase post SCI significantly reduced secondary injury-induced tissue necrosis and improved functional performance in rats.

Rapid sciatic nerve regeneration of rats by a surface modified collagen-chitosan scaffold.

In the previous study, we attempted to use a collagen-chitosan (CCH) scaffold to mimic the bio-functional peripheral nerve and to bridge sciatic nerve defects in rats. The results demonstrated that it could support and guide the nerve regeneration after three months. In the current study, a type of peptide which carried RGD sequences was connected to the CCH surface by a chemical method. After this process, the microtubule structure of the scaffold was not changed. Then the coated scaffolds were used to repair a 15mm sciatic nerve defect in rats. Four weeks after implantation, linear growth of axons in the longitudinal structure was observed, and the number of regenerated axons remarkably increased. Two months later, the scaffold was partly absorbed and replaced by large quantity of regenerated axons. Importantly, the functional examinations also support the morphological results. Compared with the CCH group, all of the achievements revealed the superior function of RGD-CCH in the rapid regeneration of injured sciatic nerve.

Comparative analysis of protein expression in differentiated thyroid tumours: a multicentre study.

This study compared clinical features and protein expression profiles in differentiated thyroid tumours to identify protein markers with the potential for indicating malignancy status. Tissue microarrays were constructed using 119 thyroid tumour samples (45 papillary carcinomas, 26 follicular carcinomas, 48 adenomas). Generally, there was overexpression of proliferating cell nuclear antigen (PCNA), p53, matrix metalloproteinase (MMP)-7, Hector Battifora mesothelial-1 (HBME-1), MMP-2, pituitary tumour-transforming gene (PTTG) and human telomerase reverse transcriptase (hTERT) in malignant thyroid carcinomas, and overexpression of fragile histidine triad (FHIT), p16 and E-cadherin in thyroid adenomas. Multiple factor binary logistic regression analysis indicated that MMP-2, HBME-1, p16 and FHIT were independently related to differentiated thyroid tumours. Receiver-operating characteristics for these four factors showed HBME-1 as best for diagnostic accuracy. Sensitivity and specificity were enhanced using an HBME-1 and p16 cluster. HBME-1 expression was not significantly different for papillary and follicular carcinomas, whereas p16 expression was significantly specific.

In-situ engineering of cartilage repair: a pre-clinical in-vivo exploration of a novel system.

This investigation explores a new cartilage repair technique that uses a novel method to secure a non-woven multifilamentous scaffold in the defect site after microfracture. The hypothesis is that a scaffold provides a larger surface area for attachment and proliferation of the mesenchymal stem cells that migrate from the bone marrow. Two in-vivo studies were undertaken in an ovine model. The first study, which lasted for 8 weeks, aimed to compare the new technique with microfracture. Chondral defects, 7 mm in diameter, were created in both femoral medial condyles of five ewes. One defect was treated with the new technique while the contralateral knee was treated with microfracture alone. The results revealed that the quantity of repair tissue was significantly greater in the defects treated with the new system. The second study had two time points, 3 and 6 months, and used 13 ewes. In this study, both defects were treated with the new technique but one received additional subchondral drilling in order to stimulate extra tissue growth. The majority of the implants had good tissue induction, filling 50-100 per cent of the defect volume, while the compressive modulus of the repairs was in the range of 40-70 per cent of that for the surrounding cartilage. In addition, hyaline-like cartilage was seen in all the repairs which had the additional drilling of the subchondral bone.

Light and low-frequency pulsatile hydrostatic pressure enhances extracellular matrix formation by bone marrow mesenchymal cells: an in-vitro study with special reference to cartilage repair.

Ovine bone marrow mesenchymal cells (BMMCs) were seeded on to non-woven filamentous plasma-treated polyester scaffolds and cultured in a chondrogenic medium for 4 weeks. Thereafter a pulsatile hydrostatic pressure (PHP) was applied to these cell-scaffolds constructs at an amplitude of 0.1 MPa and frequency of 0.25 Hz, for 30 min a day, over a period of 10 days. Samples (n = 6) were removed 24 h after PHP stimulation at days 1, 4, 7, and 10 for biochemical analysis. Similar analyses were conducted, at the same time points, on control samples that were not subjected to a PHP. The results showed that the glycosaminoglycan (GAG) content did not significantly increase until after the application of a PHP for 7 days. The GAG content was 1.5 and 2.7 times higher in the PHP group than in the control group at days 7 and 10 respectively (p<0.01). The deoxyribonucleic acid (DNA) content was 1.5 times greater in the PHP group than in the control group at day 10 (p<0.01). GAG synthesis amounts, expressed as the total GAG contents per microgram of DNA, were 1.6 and 1.8 times higher in the PHP group than in the control group at days 7 and 10 respectively (p<0.01). The total collagen content in the medium did not change until after PHP application for 10 days, when it was 1.9 times higher than the control (p < 0.05). The results suggest that a light PHP applied at a low frequency has a cumulative stimulatory effect on the BMMCs' metabolic activities including cell proliferation and synthesis of the extracellular matrix.

The role of XPB in cell apoptosis and viability and its relationship with p53, p21(waf1/cip1) and c-myc in hepatoma cells.

BACKGROUND: Accumulation of DNA damage has been implicated in hepatocarcinogenesis. XPB plays a pivotal part in repairing damaged DNA. However, up to now, the biological effect of XPB on hepatoma cells remains elusive. MATERIALS AND METHODS: Here, we investigated the role of XPB in the apoptosis and the viability of hepatoma cells by using the terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end-labelling and cell viability assay; we also investigated their relationship with p53, p21(waf1/cip1) and c-myc by using the RT-PCR and Western blot. RESULTS: Compared with the control cells HepG2/pcDNA3.1 or HepG2, XPB-transfected HepG2 cells (HepG2/pcDNA3.1-XPB) displayed lower viability, weaker activity and higher apoptosis index. At the same time, an increased expression of p21(waf1/cip1) mRNA, protein and p53 protein in addition to a decreased expression of c-myc mRNA and protein were detected in HepG2/pcDNA3.1-XPB cells. CONCLUSIONS: Our results indicated that XPB could inhibit the proliferation of hepatoma cells and had a positive effect on the expression of p53 and p21(waf1/cip1) but a negative effect on c-myc.

Vascular endothelial growth factor gene therapy with intramuscular injections of plasmid DNA enhances the survival of random pattern flaps in a rat model.

The objective of this study was to determine the effects of the naked plasmid DNA encoding vascular endothelial growth factor (VEGF) on the survival of random flaps on rats. Thirty Sprague-Dawley rats whose random flaps were elevated on the back were randomised into three groups of 10 animals each. In the experimental group, the naked plasmid DNA encoding VEGF was injected directly into the panniculus carnosus of the flap. In the two control groups, either control plasmid DNA or physiologic saline was injected. After 7 days, the flaps were evaluated with the following devices: RT-PCR for the expression of VEGF gene, immunohistochemistry for the expression of VEGF protein, histology for vascular density, single photon emission computerised tomography for RBC in the flap, and image analysis for flap survival area. Notably increased expressions of VEGF mRNA and VEGF protein were found in the treatment group. Vascular density was markedly more increased in the treatment group than those in the two control groups (P < 0.01). Compared with the two control groups, the flap treated with VEGF plasmid DNA showed a more significantly enhanced tissue viability: 87 +/- 5 versus 47 +/- 6% for the control plasmid DNA group and 46 +/- 5% for the saline group (P < 0.01). Our results indicated that the VEGF gene therapy was able to enhance the survival of random pattern flaps by inducing angiogenesis.

Effects of nonenzymatic glycosylation of extracellular matrix components on cell survival and sensory neurite extension in cell culture.

Diabetic sensory polyneuropathy is characterized by a distal axonopathy of dying-back type. It is accompanied by a failure of axonal regeneration, in which nonenzymatic glycosylation (glycation) of the extracellular matrix may be involved. In the present study, the effects of glycation of collagen IV and laminin, major components of basal lamina, on neuron survival and neurite extension were investigated in tissue culture. Fast glycation of laminin was achieved by incubation with glycolaldehyde and glycation of collagen IV by incubation with glucose. The degree of glycation was estimated by fluorescence analysis. Glycated or nonglycated laminin or collagen IV were used as substrates for culture of dorsal root ganglion (DRG) neurons from neonatal rats. Cultures were assessed for the proportion of cells attaching to the substrate, surviving and bearing neurites. Cell attachment and the proportion bearing neurites were significantly reduced on collagen IV glycated for 2 weeks, but survival was only affected by glycation for 4 or 5 weeks. All 3 parameters were significantly reduced on glycated compared with unglycated laminin. Glycation of both laminin and collagen IV produced considerable morphological differences in the cultured neurons on scanning electron microscopy. Dissociated DRG neurons from adult animals with streptozotocin-induced diabetes cultured on nonglycated substrates survived less well and produced fewer neurites. Glycation of collagen IV and laminin thus affects neuronal survival, neurite production and cell morphology, and diabetes affects both the survival of sensory neurons in culture and their ability to extend neurites.

Calyculin A-induced vimentin phosphorylation sequesters 14-3-3 and displaces other 14-3-3 partners in vivo.

14-3-3 proteins bind their targets through a specific serine/threonine-phosphorylated motif present on the target protein. This binding is a crucial step in the phosphorylation-dependent regulation of various key proteins involved in signal transduction and cell cycle control. We report that treatment of COS-7 cells with the phosphatase inhibitor calyculin A induces association of 14-3-3 with a 55-kDa protein, identified as the intermediate filament protein vimentin. Association of vimentin with 14-3-3 depends on vimentin phosphorylation and requires the phosphopeptide-binding domain of 14-3-3. The region necessary for binding to 14-3-3 is confined to the vimentin amino-terminal head domain (amino acids 1-96). Monomeric forms of 14-3-3 do not bind vimentin in vivo or in vitro, indicating that a stable complex requires the binding of a 14-3-3 dimer to two sites on a single vimentin polypeptide. The calyculin A-induced association of vimentin with 14-3-3 in vivo results in the displacement of most other 14-3-3 partners, including the protooncogene Raf, which nevertheless remain capable of binding 14-3-3 in vitro. Concomitant with 14-3-3 displacement, calyculin A treatment blocks Raf activation by EGF; however, this inhibition is completely overcome by 14-3-3 overexpression in vivo or by the addition of prokaryotic recombinant 14-3-3 in vitro. Thus, phosphovimentin, by sequestering 14-3-3 and limiting its availability to other target proteins can affect intracellular signaling processes that require 14-3-3.

A method for isolation and purification of nematode larvae.

A new apparatus for isolating, purifying, and collecting larvae of nematodes is described. Results of collecting larvae with Baermann's traditional method and the pasteur pipette cotton plug method are compared to those obtained with the device in this research note. It is simple, easy to use, and a more effective apparatus for the isolation, purification, and collection of nematode larvae.

Analysis of the mode of action of a novel immunosuppressant FTY720 in mice.

Accelerated lymphocyte homing and apoptosis have been suggested to contribute to potent immunosuppressive effects of FTY720, however, its main mechanism of action remains to be fully elucidated. Here, we examined the mode of action of FTY720 in mice. FTY720, when given at a single dose of 1 mg/kg, markedly decreased the number of peripheral blood lymphocytes (PBL) but moderately increased the lymphocyte numbers in lymph nodes (LN) and Peyer's patches (PP) in normal mice, as previously observed in rats. However, the sharp decrease in PBL numbers was also observed in aly/aly mice lacking LN and PP, indicating that this phenomenon is not explained by accelerated lymphocyte homing to LN and PP. In addition, the finding that a single administration of FTY720 did not suppress proliferative responses of T cells suggested that the PBL reduction could occur without inhibiting lymphocyte functions. However, when administered at the same dose for 2 weeks, FTY720 induced severe systemic lymphopenia, as well as marked suppression of lymphocyte proliferative responses in normal mice. The same treatment also prolonged skin allograft survival in aly/aly mice. Our results suggest that FTY720 suppresses in vivo immune functions mainly by inducing systemic lymphopenia and also by inhibiting T cell functions.

Detection of circulating antigens and antibodies in Toxocara canis infection among children in Chengdu, China.

In order to determine the seroprevalence of Toxocara spp. infection in children from Chengdu, we performed an enzyme-linked immunosorbent assay (ELISA) and sandwich ELISA (S-ELISA) with excretory-secretory antigens isolated from second-stage larvae of Toxocara canis (TES-Ag ELISA). The seroprevalences of T. canis antibodies in the children from rural areas, urban districts, and urban districts with recent Ascaris lumbricoides infection were 17.7% (59/333), 2.1% (4/186), and 2.6% (1/38), respectively. Among 63 suspected patients with symptoms of T. canis infection, 31 had positive antibodies. The inhibition assay showed an apparent inhibiting capacity of TES-Ag for the antibody against T. canis larvae. The result of S-ELISA demonstrated that circulating antigens of T. canis larvae could be detected in part of the serum with positive antibodies and that the detection rate for circulating antigens in the sera could be improved by polyethylene glycol-acid treatment. This is the first epidemiological study to confirm the existence of T. canis infection and Toxocara-larvae migrans in Chengdu by the combination of TES-Ag ELISA and S-ELISA.

Selective reinnervation of regenerating mixed nerve fibres across a silicone tube gap. Further experimental evidence of neurotropism.

This study investigated specific regeneration of a mixed motor and sensory nerve by the method of spinal dorsal root ganglions resection. A 10 mm segment of tibial nerve was resected and the nerve ends inserted in a silicone tube. Fourteen weeks later, dorsal root ganglia from L6 to S1 were resected on the experiment side. Twenty weeks later, the regenerating motor nerve fibres of mixed nerves selectively grew into motor branches. The rate of misdirected growth in mixed nerves was less than 6%. These results suggest that regenerating motor and sensory axons of mixed nerves are able to select their distal target organs accurately. Better results may be obtained using the entubulation repair method.

Identification of the 14.3.3 zeta domains important for self-association and Raf binding.

The 14.3.3 zeta protein is a ubiquitous and abundant arachidonate-selective acyltransferase and putative phospholipase A2, which self-assembles into dimers and binds to c-Raf-1 and other polypeptides in vitro and in intact cells. The 14.3.3 polypeptides endogenous to Sf9 cells associate in situ with both active and inactive recombinant Raf and copurify at a fairly reproducible molar ratio that is probably 1. Purified baculoviral recombinant Raf, despite its preassociated 14.3.3 polypeptide, binds additional recombinant 14.3.3 zeta polypeptide in vitro, in a saturable and specific reaction, forming a complex that is resistant to 1 M LiCl. A two-hybrid analysis indicates that 14.3.3 zeta binds primarily to Raf noncatalytic sequences distinct from those that bind Ras-GTP, and in vitro 14.3.3 zeta binds to Raf without inhibiting the Ras-Raf association or Raf-catalyzed MEK phosphorylation. Deletion analysis of 14.3.3 zeta (1-245) indicates that the 14.3.3 domain responsible for binding to Raf extends over the carboxyl-terminal 100 amino acids, whereas 14.3.3 dimerization is mediated by amino-terminal sequences. As with Ras, the 14.3.3 zeta polypeptide does not activate purified Raf directly in vitro. Moreover, expression of recombinant 14.3.3 zeta in COS cells beyond the substantial level of endogenous 14.3.3 protein does not alter endogenous Raf kinase, as judged by the activity of a cotransfected Erk-1 reporter. Coexpression of recombinant 14.3.3 with recombinant Myc-tagged Raf in COS cells does increase substantially the Myc-Raf kinase activity achieved during transient expression, which is attributable primarily to an increased level of Myc-Raf polypeptide, without alteration of Myc-Raf specific activity or the activation that occurs in response to epidermal growth factor or 12-O-tetradecanoylphorbol-13-acetate. Nevertheless, evidence that 14.3.3 actively participates in Raf activation in situ is provided by the finding that although full-length 14.3.3 zeta binds active Raf in situ, truncated versions of 14.3.3, some of which bind Raf polypeptide in situ nearly as well as full-length 14.3.3 zeta, are recovered in association only with inactive Raf polypeptides. Thus, 14.3.3 polypeptides bind tightly to one or more sites on c-Raf. Overexpression of 14.3.3 zeta enhances the expression of recombinant Raf, perhaps by stabilizing the Raf polypeptide. In addition, Raf polypeptides bound to truncated 14.3.3 polypeptides are unable to undergo activation in situ, indicating that 14.3.3 participates in the process of Raf activation by mechanisms that remain to be elucidated.