A novel intranuclear RNA vector system for long-term stem cell modification.

Genetically modified stem and progenitor cells have emerged as a promising regenerative platform in the treatment of genetic and degenerative disorders, highlighted by their successful therapeutic use in inherent immunodeficiencies. However, biosafety concerns over insertional mutagenesis resulting from integrating recombinant viral vectors have overshadowed the widespread clinical applications of genetically modified stem cells. Here, we report an RNA-based episomal vector system, amenable for long-term transgene expression in stem cells. Specifically, we used a unique intranuclear RNA virus, borna disease virus (BDV), as the gene transfer vehicle, capable of persistent infections in various cell types. BDV-based vectors allowed for long-term transgene expression in mesenchymal stem cells (MSCs) without affecting cellular morphology, cell surface CD105 expression or the adipogenicity of MSCs. Similarly, replication-defective BDV vectors achieved long-term transduction of human induced pluripotent stem cells, while maintaining the ability to differentiate into three embryonic germ layers. Thus, the BDV-based vectors offer a genomic modification-free, episomal RNA delivery system for sustained stem cell transduction.

Revision of anterior cruciate ligament reconstruction with allografts in patients younger than 40 years old: a 2 to 4 year results.

PURPOSE: The purpose of this study is first to report the outcomes, at 4 years follow-up, in revision ACL surgery using allografts in patients younger than 40 years old, and then compared soft tissue allografts to bone tendon allografts. METHODS: This retrospective study included 47 patients who underwent ACL revision surgery with fresh-frozen allografts. Patellar tendon allograft or tibialis anterior allograft was used. Twenty-seven patients undergoing ACL revision with patellar tendon allograft were compared retrospectively with twenty-two patients undergoing the same procedure with soft tissue tibialis anterior allograft. Lysholm, IKDC, and KT-1000 values were obtained preoperatively and postoperatively. RESULTS: The average patient follow-up was 4.6 years (±2.5). The mean age at time of the revision was 34 years old (±6.3). Overall, patients reported the overall condition of their knee as excellent or good in 85% of the patients (10 excellent, 33 good). Based on their experience, 85% would have the surgery again if they had the same problem in the other knee. Both subgroups experienced significant improvement in Lysholm, IKDC, and KT-1000 values, with no difference found between groups at final follow-up. CONCLUSION: Revision ACL with allografts has excellent and good results in 85% of patients younger than 40 years old. No statistical difference was seen between soft tissue (tibialis anterior) and patellar tendon allograft. LEVEL OF EVIDENCE: IV.

Mitochondrial fragmentation and superoxide anion production in coronary endothelial cells from a mouse model of type 1 diabetes.

AIMS/HYPOTHESIS: Mitochondria frequently change their shapes by fusion and fission and these morphological dynamics play important roles in mitochondrial function and development as well as programmed cell death. The goal of this study is to investigate whether: (1) mitochondria in mouse coronary endothelial cells (MCECs) isolated from diabetic mice exhibit increased fragmentation; and (2) chronic treatment with a superoxide anion (O(2)(-)) scavenger has a beneficial effect on mitochondrial fragmentation in MCECs. METHODS: MCECs were freshly isolated and lysed for protein measurement, or cultured to determine mitochondrial morphology and O(2)(-) production. For the ex vivo hyperglycaemia experiments, human coronary endothelial cells were used. RESULTS: Elongated mitochondrial tubules were observed in MCECs isolated from control mice, whereas mitochondria in MCECs from diabetic mice exhibited augmented fragmentation. The level of optic atrophy 1 (OPA1) protein, which leads to mitochondrial fusion, was significantly decreased, while dynamin-related protein 1 (DRP1), which leads to mitochondrial fission, was significantly increased in MCECs from diabetic mice. Diabetic MCECs exhibited significantly higher O(2)(-) concentrations in cytosol and mitochondria than control MCECs. Administration of the O(2)(-) scavenger TEMPOL to diabetic mice for 4 weeks led to a significant decrease in mitochondrial fragmentation without altering the levels of OPA1 and DRP1 proteins in MCECs. High-glucose treatment for 24 h significantly induced mitochondrial fragmentation, which was restored by TEMPOL treatment. In addition, excess O(2)(-) production, either in cytosol or in mitochondria, significantly increased mitochondrial fragmentation. CONCLUSIONS/INTERPRETATION: These data suggest that lowering the O(2)(-) concentration can restore the morphological change in mitochondria and may help improve mitochondrial function in diabetic MCECs.

Emerging and reemerging influenza virus infections.

Influenza A virus infection occurs in many species. Wild waterfowl harbor the widest variety of influenza A viruses and serve as a constant reservoir for the emergence of new viruses. Highly pathogenic avian influenza, or "fowl plague," has been a known poultry disease for more than 130 years. It continues to emerge and reemerge, but global changes in trade and poultry production have expanded the impact and geographic range of these outbreaks. One subtype of highly pathogenic avian influenza, H5N1, has infected poultry on several continents as well as many people, leading to a human disease that is markedly different from seasonal influenza and that is associated with high mortality.

Composite adenocarcinoma and large cell neuroendocrine carcinoma of the rectum.

Composite glandular-endocrine tumors of the gastrointestinal tract are rare neoplasms. Even more uncommon are the so-called amphicrine tumors, lesions in which dual epithelial and endocrine differentiation occurs in the same cell. We describe a patient who complained of rectal pain and bleeding with a mixed or composite adenocarcinoma and neuroendocrine carcinoma of the rectum. Histological examination revealed a distinct adenocarcinoma of conventional type with glandular structures admixed intimately with a neuroendocrine carcinoma. The latter component was deeply infiltrative, while the adenocarcinoma occupied the more superficial aspect of the tumor. What was interesting was the immunophenotype of the lesion: cytokeratin (CK) 20 expression was very focal in the adenocarcinoma component and negative in the neuroendocrine carcinoma, while CK 7 was expressed strongly in the adenocarcinoma and only focally in the neuroendocrine component. This cytokeratin profile suggests a possible origin from the anal transitional zone.

Responses of Ribulose-1,5-Bisphosphate Carboxylase, Cytochrome f, and Sucrose Synthesis Enzymes in Rice Leaves to Leaf Nitrogen and Their Relationships to Photosynthesis.

The photosynthetic gas-exchange rates and various biochemical components of photosynthesis, including ribulose-1,5-bisphosphate carboxylase (Rubisco) content, cytochrome (Cyt) f content, and the activities of two sucrose synthesis enzymes, were examined in young, fully expanded leaves of rice (Oryza sativa L.) grown hydroponically in different nitrogen concentrations. The light-saturated rate of photosynthesis at an intercellular CO2 pressure of 20 Pa (CO2-limited photosynthesis) was linearly dependent on leaf nitrogen content, but curvilinearly correlated with Rubisco content. This difference was due to a greater than proportional increase in Rubisco content relative to leaf nitrogen content and the presence of a CO2 transfer resistance between the intercellular air spaces and the carboxylation sites. CO2-limited photosynthesis was proportional to Cyt f content, one of the key components of electron transport, but was not proportional to the activities of cytosolic fructose-1,6-bisphosphatase and sucrose phosphate synthase, the two regulatory enzymes of sucrose synthesis. Light-saturated photosynthesis above an intercellular CO2 pressure of 60 Pa (CO2-saturated photosynthesis) was curvilinearly dependent on leaf nitrogen content. This CO2-saturated photosynthesis was proportional to Cyt f content in the low- and normal-nitrogen leaves, and correlated better with the activities of cytosolic fructose-1,6-bisphosphatase and sucrose phosphate synthase in the high-nitrogen leaves. The increase in the activities of these two enzymes with increasing leaf nitrogen was not as great as the increase in Cyt f content. Thus, as leaf nitrogen increased, the limitation caused by the activities of sucrose synthesis enzymes came into play, which resulted in the curvilinear relationship. However, this limitation by sucrose synthesis enzymes did not affect photosynthesis under normal ambient air.

Effects of Growth Temperature on the Responses of Ribulose-1,5-Biphosphate Carboxylase, Electron Transport Components, and Sucrose Synthesis Enzymes to Leaf Nitrogen in Rice, and Their Relationships to Photosynthesis.

Effects of growth temperature on the photosynthetic gas-exchange rates and their underlying biochemical properties were examined in young, fully expanded leaves of rice (Oryza sativa L.). The plants were grown hydroponically under day/night temperature regimes of 18/15[deg]C, 23/18[deg]C, and 30/23[deg]C and all photosynthetic measurements were made at a leaf temperature of 25[deg]C and an irradiance of 1800 [mu]mol quanta m-2 s-1. Growth temperature affected the photosynthetic CO2 response curve. The relative ratio of the initial slope to the CO2-saturated photosynthesis increased with rising growth temperature. This was caused mainly by an increase in CO2-limited photosynthesis for a given leaf nitrogen content with rising growth temperature. However, there was no difference in ribulose-1,5-bisphosphate carboxylase (Rubisco) content at any given leaf nitrogen content among temperature treatments. In addition, the activation state and catalytic turnover rate of Rubisco were not affected by growth temperature. The increase in CO2-limited photosynthesis with rising growth temperature was the result of an increase in the CO2 transfer conductance between the intercellular airspaces and the carboxylation sites. The amounts of total chlorophyll and light-harvesting chlorophyll a/b protein II increased for the same leaf nitrogen content with rising growth temperature, but the amounts of cytochrome f and coupling factor 1 and the activities of cytosolic fructose-1,6-bisphosphatase and sucrose-phosphate synthase were the same between plants grown at 23/18[deg]C and those grown at 30/23[deg]C. Similarly, CO2-saturated photosynthesis was not different for the same leaf nitrogen content between these treatments. For the 18/15[deg]C-grown plants, a slight decrease in the amounts of cytochrome f and coupling factor 1 and an increase in the activities of cytosolic fructose-1,6-bisphosphatase and sucrose-phosphate synthase were found, but these were not reflected in CO2-saturated photosynthesis.

The Effect of Elevated Partial Pressures of CO2 on the Relationship between Photosynthetic Capacity and N Content in Rice Leaves.

The effects of growth CO2 levels on the photosynthetic rates; the amounts of ribulose-1,5-bisphosphate carboxylase (Rubisco), chlorophyll (Chl), and cytochrome f; sucrose phosphate synthase activity; and total N content were examined in young, fully expanded leaves of rice (Oryza sativa L.). The plants were grown hydroponically under two CO2 partial pressures of 36 and 100 Pa at three N concentrations. The light-saturated photosynthesis at 36 Pa CO2 was lower in the plants grown in 100 Pa CO2 than those grown in 36 Pa CO2. Similarly, the amounts of Rubisco, Chl, and total N were decreased in the leaves of the plants grown in 100 Pa CO2. However, regression analysis showed no differences between the two CO2 treatments in the relationship between photosynthesis and total N or in the relationship between Rubisco and Chl and total N. Although a relative decrease in Rubisco to cytochrome f or sucrose phosphate synthase was found in the plants grown in 100 Pa CO2, this was the result of a decrease in total N content by CO2 enrichment. The activation state of Rubisco was also unaffected by growth CO2 levels. Thus, decreases in the photosynthetic capacity of the plants grown in 100 Pa CO2 could be simply accounted for by a decrease in the absolute amount of leaf N.

Effects of Two Strains of Tobacco Mosaic Virus on Photosynthetic Characteristics and Nitrogen Partitioning in Leaves of Nicotiana tabacum cv Xanthi during Photoacclimation under Two Nitrogen Nutrition Regimes.

Photoacclimation was studied in tobacco leaves (Nicotiana tabacum cv Xanthi) infected with two strains of tobacco mosaic virus (TMV) and grown under different light and nitrogen nutrition regimes. Photosynthetic acclimation measured by the quantum yield and the maximum rate in saturating light of CO2-saturated photosynthesis was impaired to a greater extent in tobacco leaves infected with TMV strain PV230 than in those infected with TMV strain PV42. Infection with TMV strain PV230 severely impaired photosynthetic acclimation at high light/low nitrogen and during transfer from low to high light. Expanding leaves showing chlorotic-mosaic symptoms had greatly reduced capacity to acclimate to high light compared with controls and with developed leaves without visible symptoms. We conclude that the failure of expanding leaves to acclimate was largely due to the destruction of chloroplasts in yellow areas of the tissue, accompanied by severe reduction in ribulose-1,5-bisphosphate carboxylase/oxygenase levels, and corresponding reduction in photosynthesis on a leaf-area basis. When corrected for areas of healthy green tissue, photoacclimation of infected leaves was the same as that of controls. Visible symptom development was greatest in high light/low nitrogen treatments. In developed leaves without visible symptoms, virus accumulation, which was as extensive as in expanding leaves, accelerated senescence and impaired photoacclimation during transfer from low light to high light. Generally, infection with TMV strain PV42 did not impair photosynthetic acclimation and even enhanced it in some treatments, even though virus accumulated to the same concentration as in PV230-infected leaves. These data show that TMV does not simply impair photoacclimation in tobacco by competing with chloroplasts for leaf nitrogen reserves. Rather, specific properties of severe strains, such as PV230, which lead to visible symptom development and patchy loss of photosynthetic activity in expanding leaves as well as general acceleration of chloroplast senescence in developed leaves, contribute to impaired photoacclimation, which is generally exacerbated by low nitrogen nutrition.

Growth and N Allocation in Rice Plants under CO2 Enrichment.

The effects of CO2 enrichment on growth and N allocation of rice (Oryza sativa L.) were examined. The plants were grown hydroponically in growth chambers with a 14-h photoperiod (1000 [mu]mol quanta m-2 s-1) and a day/night temperature of 25/20[deg]C. From the 28th to 70th d after germination, the plants were exposed to two CO2 partial pressures, namely 36 and 100 Pa. The CO2 enrichment increased the final biomass, but this was caused by a stimulation of the growth rate during the first week of the exposure to elevated CO2 partial pressures. The disappearance of the initial stimulation of the growth rate was associated with a decreased leaf area ratio. Furthermore, CO2 enrichment decreased the investment of N in the leaf blades, whereas the N allocation into the leaf sheaths and roots increased. Thus, the decrease in leaf N content by CO2 enrichment was not due to dilution of N caused by a relative increase in the plant biomass but was due to the change in N allocation at the whole-plant level. We conclude that the growth responses of rice to CO2 enrichment are mainly controlled by leaf area expansion and N allocation into leaf blades at the whole-plant level.

Does Decrease in Ribulose-1,5-Bisphosphate Carboxylase by Antisense RbcS Lead to a Higher N-Use Efficiency of Photosynthesis under Conditions of Saturating CO2 and Light in Rice Plants?

Rice (Oryza sativa L.) plants with decreased ribulose-1,5-bisphosphate carboxylase (Rubisco) were obtained by transformation with the rice rbcS antisense gene under the control of the rice rbcS promoter. The primary transformants were screened for the Rubisco to leaf N ratio, and the transformant with 65% wild-type Rubisco was selected as a plant set with optimal Rubisco content at saturating CO2 partial pressures for photosynthesis under conditions of high irradiance and 25[deg]C. This optimal Rubisco content was estimated from the amounts and kinetic constants of Rubisco and the gas-exchange data. The R1 selfed progeny of the selected transformant were grown hydroponically with different N concentrations. Rubisco content in the R1 population was distributed into two groups: 56 plants had about 65% wild-type Rubisco, whereas 23 plants were very similar to the wild type. Although the plants with decreased Rubisco showed 20% lower rates of light-saturated photosynthesis in normal air (36 Pa CO2), they had 5 to 15% higher rates of photosynthesis in elevated partial pressures of CO2, (100-115 Pa CO2) than the wild-type plants for a given leaf N content. We conclude that the rice plants with 65% wild-type Rubisco show a higher N-use efficiency of photosynthesis under conditions of saturating CO2 and high irradiance.

Salt-inducible kinase is involved in the ACTH/cAMP-dependent protein kinase signaling in Y1 mouse adrenocortical tumor cells.

The involvement of salt-inducible kinase, a recently cloned protein serine/threonine kinase, in adrenal steroidogenesis was investigated. When Y1 mouse adrenocortical tumor cells were stimulated by ACTH, the cellular content of salt-inducible kinase mRNA, protein, and enzyme activity changed rapidly. Its level reached the highest point in 1-2 h and returned to the initial level after 8 h. The mRNA levels of cholesterol side-chain cleavage cytochrome P450 and steroidogenic acute regulatory protein, on the other hand, began to rise after a few hours, reaching the highest levels after 8 h. The salt-inducible kinase mRNA level in ACTH-, forskolin-, or 8-bromo-cAMP-treated Kin-7 cells, mutant Y1 with less cAMP-dependent PKA activity, remained low. However, Kin-7 cells, when transfected with a PKA expression vector, expressed salt-inducible kinase mRNA. Y1 cells that overexpressed salt-inducible kinase were isolated, and the mRNA levels of steroidogenic genes in these cells were compared with those in the parent Y1. The level of cholesterol side-chain cleavage cytochrome P450 mRNA in the salt-inducible kinase-overexpressing cells was markedly low compared with that in the parent, while the levels of Ad4BP/steroidogenic factor-1-, ACTH receptor-, and steroidogenic acute regulatory protein-mRNAs in the former were similar to those in the latter. The ACTH-dependent expression of cholesterol side-chain cleavage cytochrome P450- and steroidogenic acute regulatory protein-mRNAs in the salt-inducible kinase-overexpressing cells was significantly repressed. The promoter activity of the cholesterol side-chain cleavage cytochrome P450 gene was assayed by using Y1 cells transfected with a human cholesterol side-chain cleavage cytochrome P450 promoter-linked reporter gene. Addition of forskolin to the culture medium enhanced the cholesterol side-chain cleavage cytochrome P450 promoter activity, but the forskolin-dependently activated promoter activity was inhibited when the cells were transfected with a salt-inducible kinase expression vector. This inhibition did not occur when the cells were transfected with a salt-inducible kinase (K56M) vector that encoded an inactive kinase. The salt-inducible kinase's inhibitory effect was also observed when nonsteroidogenic, nonAd4BP/steroidogenic factor-1 -expressing, NIH3T3 cells were used for the promoter assays. These results suggested that salt-inducible kinase might play an important role(s) in the cAMP-dependent, but Ad4BP/steroidogenic factor-1-independent, gene expression of cholesterol side-chain cleavage cytochrome P450 in adrenocortical cells.

A comparative study on the rat aorta and mesenteric arterial bed of the possible role of nitric oxide in the desensitization of the vasoconstrictor response to an alpha 1-adrenoceptor agonist.

1. In thoracic aortic strips with intact endothelium, the first and second (1 h later) dose-response curves obtained with methoxamine were almost the same. 2. Methoxamine caused a dose-dependent increase in perfusion pressure in the rat isolated mesenteric arterial bed, but the second (1 h later) dose-response curve for methoxamine showed a significant attenuation of the response in comparison with the first. 3. The attenuation shown by the second dose-response curve for methoxamine was significantly reduced, but not abolished, in mesenteric arterial beds without endothelium. Incubating endothelium-intact mesenteric arterial beds with NG-nitro-L-arginine (L-NOARG) caused a significant, but not complete, reversal of the attenuation shown in the second dose-response curve. 4. Incubating the mesenteric arterial bed with capsaicin, tetrodotoxin, indomethacin or with isotonic high k+ (60 mM) plus nicardipine did not affect the above attenuation seen in the second dose-response curve. 5. The guanosine 3':5'-cyclic monophosphate (cyclic GMP) level in the effluent from the perfused mesenteric arterial bed was significantly increased after the second exposure to methoxamine. This effect was significantly smaller after removal of the endothelium or pretreatment with L-NOARG. 6. These results suggest that a desensitization to methoxamine develops rapidly in the mesenteric arterial bed, but not in the aorta, and that release of nitric oxide from the endothelium plays a major role in this desensitization.

Elevated plasma endothelin-1 level in streptozotocin-induced diabetic rats and responsiveness of the mesenteric arterial bed to endothelin-1.

1. Both the plasma endothelin-1 (ET-1) levels and the plasma glucose levels were markedly elevated in streptozotocin (STZ)-induced diabetic rats. 2. The maximum contractile response of the mesenteric arterial bed to ET-1 was significantly reduced, and the vasodilatation induced by the ET(B)-receptor agonist IRL-1620 in the mesenteric arterial bed was significantly reduced in STZ-induced diabetic rats. 3. ET-1 (10(-8) M) caused a transient vasodilatation followed by a marked vasoconstriction in methoxamine-preconstricted mesenteric arterial beds. The ET-1-induced vasodilatation was significantly larger in beds from diabetic rats than in those from age-matched controls. By contrast, the ET-1-induced vasoconstriction was significantly smaller in STZ-induced diabetic rats than in the controls. 4. Both removal of the endothelium with Triton X-100 and preincubation with BQ-788 (10(-6) M) (ET(B)-receptor antagonist) abolished the ET-1-induced vasodilatation. Preincubation with BQ-485 (10(-6) M) or BQ-123 (3 x 10(-6)) (ET(A)-receptor antagonist) significantly augmented the ET-1-induced vasodilatation in control mesenteric arterial beds, but not that in beds from diabetic rats. 5. These results demonstrate that marked increases not only in plasma glucose, but also in plasma ET-1 occur in STZ-induced diabetic rats. We suggest that the decreased contractile response and the increased vasodilator response of the mesenteric arterial bed to ET-1 may both be due to desensitization of ET(A) receptors, though ET(B) receptors may also be desensitized. This desensitization may result from the elevation of the plasma ET-1 levels seen in STZ-induced diabetic rats.

Mechanisms underlying the attenuation of endothelium-dependent vasodilatation in the mesenteric arterial bed of the streptozotocin-induced diabetic rat.

Experiments were designed to investigate the mechanisms underlying the diabetes-related impairment of the vasodilatations of the perfused mesenteric arterial bed induced by acetylcholine (ACh) and K(+). In streptozotocin (STZ)-diabetic rats, the ACh-induced endothelium-dependent vasodilatation was attenuated. The dose-response curves for ACh in control and diabetic rats were each shifted to the right by N(G)-nitro-L-arginine (L-NOARG) and by isotonic high K(+) (60 mM). The ACh dose-response curves under isotonic high K(+) were not different between control and diabetic rats. We also examined the vasodilatation induced by K(+), which is a putative endothelium-derived hyperpolarizing factor (EDHF). The mesenteric vasodilatation induced by a single administration of K(+) was greatly impaired in STZ-induced diabetic rats. Treatment with charybdotoxin plus apamin abolished the ACh-induced vasodilatation but enhanced the K(+)-induced response in controls and diabetic rats. After pretreatment with ouabain plus BaCl(2), the ACh-induced vasodilatation was significantly impaired and the K(+)-induced relaxation was abolished in both control and diabetic rats. The impairment of the endothelium-dependent vasodilatation of the mesenteric arterial bed seen in STZ-induced diabetic rats may be largely due to a defective vascular response to EDHF. It is further suggested that K(+) is one of the endothelium-derived hyperpolarizing factors and that the vasodilatation response to K(+) is impaired in the mesenteric arterial bed from diabetic rats.

Mechanisms underlying increased release of endothelin-1 from aorta in diabetic rats.

To clarify the mechanism underlying increased endothelin-1 release in diabetic rats, we examined its release from thoracic aortas obtained from streptozotocin-induced diabetic rats. The methoxamine-induced contraction was significantly inhibited by BQ-123 plus BQ-788 (specific antagonists for ET(A) and ET(B) receptors) in diabetic, but not control rats. Preincubation with phosphoramidon also inhibited the methoxamine-induced contraction in diabetic but not control rats. The expression of prepro endothelin-1 mRNA was significantly enhanced in aortas from streptozotocin-induced diabetic rats. These results suggest that the increases in the basal and alpha-agonist-induced release of endothelin-1 in the diabetic state may be due to an overexpression of the mRNA for prepro endothelin-1.

Selective reinnervation: a comparison of recovery following microsuture and conduit nerve repair.

Selective reinnervation was studied by comparing the regeneration across a conventional neurorraphy versus a conduit nerve repair. Lewis rats underwent right sciatic nerve transection followed by one of four different nerve repairs (n = 8/group). In groups I and II a conventional neurorraphy was performed and in groups III and IV the proximal and distal stumps were coapted by use of a silicone conduit with an interstump gap of 5 mm. The proximal and distal stumps in groups I and III were aligned anatomically correct and the proximal stump was rotated 180 degrees in groups II and IV (i.e. proximal peroneal nerve opposite the distal tibial nerve and the proximal tibial nerve opposite the distal peroneal nerve). By 14 weeks, there was an equivalent, but incomplete return in sciatic function index (SFI) in groups I, III, and IV as measured by walking track analysis. However, the SFI became unmeasurable by 6 weeks in all group II animals. At 14 weeks, the percent innervation of the tibialis anterior and medial gastronemius muscles by the peroneal and tibial nerves respectively was estimated by selective compound muscle action potential amplitude recordings. When fascicular alignment was reversed, there was greater tibial (P = 0.02) and lesser peroneal (P = 0.005) innervation of the gastrocnemius muscle in the conduit (group IV) versus the neurorraphy (group II) group. This suggests that the gastrocnemius muscle may be selectively reinnervated by the tibial nerve. However, there was no evidence of selective reinnervation of the tibialis anterior muscle. Despite these differences, the functional recovery in both conduit repair groups (III and IV) was equivalent to a correctly aligned microsuture repair (group I) and superior to that in the incorrectly aligned microsuture repair (group II).

An improved method for culturing cerebellar Purkinje cells with differentiated dendrites under a mixed monolayer setting.

We report here a novel cell culture protocol which facilitates in vitro survival and dendritic differentiation of cerebellar Purkinje cells in a monolayer, mixed culture setting. We found that the type of culture medium is a critical factor for the maintenance of these cells. Purkinje cells present in the single cell suspension of embryonic rat cerebellum were best maintained in a medium based on Dulbecco's modified Eagle's medium (DMEM)/F-12 without the addition of known neurotrophic factors. These cells maintained in DMEM/F-12-based media displayed an approximately 2.5-3.5-fold increase in survival compared with cells maintained in the widely used Basal Medium Eagle's (BME)-based serum-free culture medium with the same supplements. This novel protocol permits not only enhanced survival but also accelerated, improved dendritic differentiation of these cells. Purkinje cells developed highly branched spiny dendrites by 14-16 days in vitro, which matches the time course of the dendritic growth of these cells in vivo. The Purkinje cells expressed metabotropic glutamate receptor 1alpha in the cell bodies and branched dendrites, and the intradendritic calcium concentration increased when trans-ACPD, a selective agonist of this receptor, was applied. This novel protocol allows the development of functional branched dendrites and therefore is useful for electrophysiological and ion-imaging studies on dendrites of Purkinje cells grown in vitro.

Tissue-typing in human massive allografts of frozen bone.

The cases of twenty-six patients who received a massive allotransplant of frozen bone, with a known degree of histocompatibility between the donor and the recipient, were studied. Twenty-two patients were followed for more than two years (range, twenty-four to ninety-two months). Twenty-three biopsies were performed in sixteen patients from nine to seventy-eight months after transplantation. No clear relationship could be established between the degree of histocompatibility of the donor and the recipient and the incorporation of the graft, probably in part due to the number of variables involved and the polymorphism of the HLA system. However, no early massive resorption of the transplant was seen in this series, in which, by the design of the protocol, no recipients had pre-existing circulating antibodies to the antigens of the donor. Two allografts showed infiltration by round cells and vascular lesions in the absence of infection, which is suggestive of an immune response against antigens from the donor. Both matched poorly with the donor for HLA antigens. The individual who had the strongest reaction was the only recipient in the series who had a massive failure of the transplant.

Cervical myelopathy secondary to ossification of the posterior longitudinal ligament in a Caucasian patient.

Cervical myelopathy consequent on ossification of posterior longitudinal ligament (OPLL) is very rare in Caucasians. A 65-year-old Anglo-Saxon woman developed progressive gait disturbance, paresthesia in both legs and urinary urge incontinence. Radiological examination showed OPLL from fifth to seventh cervical vertebral level; the dense OPLL was graphically displayed by three-dimensional computerized tomography. Medial corpectomy, C5 to C7, and removal of OPLL, with subsequent fusion C4 to T1 using a free fibula graft resulted in clinical improvement. Three dimensional computerized tomographic imaging is a valuable diagnostic procedure in OPLL.