Transplanted modified muscle progenitor cells expressing a mixture of neurotrophic factors delay disease onset and enhance survival in the SOD1 mouse model of ALS.

Neurotrophic factors (NTFs) are essential growth factor proteins that support the development, survival, and proper function of neurons. We have developed muscle progenitor cell (MPC) populations expressing brain-derived neurotrophic factor (BDNF), glial-derived neurotrophic factor (GDNF), vascular endothelial growth factor (VEGF), or insulin-like growth factor-1 (IGF-1). Transplantation of a mixture of such MPC populations (MPC-MIX) into the hind legs of SOD1 G93A transgenic mice (SOD1 mice), the commonly used model of ALS, delayed the onset of disease symptoms by 30 days and prolonged the average lifespan by 13 days. Treated mice also showed a decrease in the degeneration of neuromuscular junction and an increase in axonal survival. Cellular mechanism assays suggest a synergistic rescue effect of NTFs that involves the AKT and BAD signaling pathways. The results suggest that long-term delivery of a mixture of several NTFs by the transplantation of engineered MPC has a beneficial effect in the ALS mouse model.

Therapeutic effect of myogenic cells modified to express neurotrophic factors in a rat model of sciatic nerve injury.

Sciatic nerve injury may cause neurological deficits, particularly muscle weakness. Previous studies have shown that administration of neurotrophic factors (NTFs), naturally occurring proteins that support the development and survival of neurons, partially protected the damaged motor neuron in the injured sciatic nerve. In the current study, we have examined whether the administration of various combinations of transfected muscle progenitor cells (MPCs) populations, each expressing a single NTF (BDNF, GDNF, IGF-1 or VEGF) or conditioned media of such culture are capable of rescuing motor neurons in culture or in vivo. We have found that the mixture of conditioned media collected from cultured myogenic cells (MPCs- MIX(+)) alleviated the toxic effect of exposure of the motor neuron cell line NSC34 to hypoxic environment. Furthermore, NTFs secreting cells transplantation, protected motor neurons in a unilateral rat sciatic nerve injury model: One day after the crush, rats underwent transplantation at the lesion site with rat myogenic cells expressing one of the four NTFs; a mixture of cells expressing all four NTFs (MPCs- MIX(+)), MPCs-GFP or PBS. We found that in rats injected with MPCs- MIX(+) the motor function was markedly preserved, compared to groups injected with cells secreting a single NTF, GFP or PBS. Transplantation of the MPCs- MIX(+) significantly inhibited the degeneration of the neuromuscular junctions and enhanced the survival of the myelinated motor axons. The injection of MPCs- MIX(+) preserved the compound muscle action potential (CMAP) as was demonstrated by motor nerve conduction studies. Our findings suggest that MPCs induced to secrete several NTFs can synergistically alleviate symptoms of sciatic nerve injury and perhaps other motor neuron disorders..

A fuzzy ARTMAP based on quantitative structure-property relationships (QSPRs) for predicting aqueous solubility of organic compounds.

Quantitative structure-property relationships (QSPRs) for estimating aqueous solubility of organic compounds at 25 degrees C were developed based on a fuzzy ARTMAP and a back-propagation neural networks using a heterogeneous set of 515 organic compounds. A set of molecular descriptors, developed from PM3 semiempirical MO-theory and topological descriptors (first-, second-, third-, and fourth-order molecular connectivity indices), were used as input parameters to the neural networks. Quantum chemical input descriptors included average polarizability, dipole moment, resonance energy, exchange energy, electron-nuclear attraction energy, and nuclear-nuclear (core-core) repulsion energy. The fuzzy ARTMAP/QSPR correlated aqueous solubility (S, mol/L) for a range of -11.62 to 4.31 logS with average absolute errors of 0.02 and 0.14 logS units for the overall and validation data sets, respectively. The optimal 11-13-1 back-propagation/QSPR model was less accurate, for the same solubility range, and exhibited larger average absolute errors of 0.29 and 0.28 logS units for the overall and validation sets, respectively. The fuzzy ARTMAP-based QSPR approach was shown to be superior to other back-propagation and multiple linear regression/QSPR models for aqueous solubility of organic compounds.

Multimedia analysis of PAHs and nitro-PAH daughter products in the Los Angeles Basin.

Polycyclic aromatic hydrocarbons (PAHs) that are released into the atmosphere may have health consequences that can be compounded by their nitro-PAH atmospheric transformation products. The available literature suggests that some of the atmospheric nitro-PAH daughter products may increase the overall environmental health risk associated with PAHs. Therefore, an important issue is whether there is merit in considering atmospheric transformation products of air toxins when conducting environmental health-risk analyses. To illustrate the above issue, a comparative analysis of the potential risk that may be imposed by PAHs and their daughter products was carried out for the Los Angeles Basin. The analysis consisted of first assessing the multimedia environmental concentration of selected PAHs and nitro-PAHs using a spatial-compartmental modeling approach coupled with available monitoring data. Multimedia concentrations were then used to estimate chemical media-specific mutagenic densities as well as average daily intake from multiple pathways, followed by cancer risk for the known carcinogens among the study chemicals. The analysis revealed that mutagenic densities of the nitro-PAH daughter products can significantly exceed those of the parent PAHs. The results of this study suggest that there is merit in further investigation of the potential contribution of nitro-PAHs to the overall environmental health risk associated with airborne PAHs.

Neural network based temperature-dependent quantitative structure property relations (QSPRs) for predicting vapor pressure of hydrocarbons.

A neural network based quantitative structure-property relationship (QSPR) was developed for the vapor pressure-temperature behavior of hydrocarbons based on a data set for 274 compounds. The optimal QSPR model was developed based on a 7-29-1 back-propagation neural network architecture using valance molecular connectivity indices (1chi(v), 3chi(v), 4chi(v)), molecular weight, and temperature as input parameters. The average absolute errors in vapor pressure predictions for the test, validation, and overall data sets were 8.2% (0.036 log P units or 23.2 kPA), 9.2% (0.039 log P units or 26.8 kPA), and 10.7% (0.046 log P units or 31.1 kPA), respectively. The performance of the QSPR for temperature-dependent vapor pressure, which was developed from a simple set of molecular descriptors, displayed accuracy of better than or well within the range of other available estimation methods.

The dystrophin / utrophin homologues in Drosophila and in sea urchin.

The gene which is defective in Duchenne muscular dystrophy (DMD) is the largest known gene containing at least 79 introns, some of which are extremely large. The product of the gene in muscle, dystrophin, is a 427 kDa protein. The same gene encodes at least two additional non-muscle full length dystrophin isoforms transcribed from different promoters located in the 5'-end region of the gene, and four smaller proteins transcribed from internal promoters located further downstream, and lack important domains of dystrophin. Several other genes, encoding evolutionarily related proteins, have been identified. To study the evolution of the DMD gene and the significance of its various products, we have searched for genes encoding dystrophin-like proteins in sea urchin and in Drosophila. We previously reported on the characterization of a sea urchin gene encoding a protein which is an evolutionary homologue of Dp116, one of the small products of the mammalian DMD gene, and on the partial sequencing of a large product of the same gene. Here we describe the full-length product which shows strong structural similarity and sequence identity to human dystrophin and utrophin. We also describe a Drosophila gene closely related to the human dystrophin gene. Like the human gene, the Drosophila gene encodes at least three isoforms of full length dystrophin-like proteins (dmDLP1, dmDLP2 and dmDLP3,), regulated by different promoters located at the 5' end of the gene, and a smaller product regulated by an internal promoter (dmDp186). As in mammals, dmDp186 and the dmDLPs share the same C-terminal and cysteine-rich domains which are very similar to the corresponding domains in human dystrophin and utrophin. In addition, dmDp186 contains four of the spectrin-like repeats of the dmDLPs and a unique N-terminal region of 512 amino acids encoded by a single exon. The full length products and the small product have distinct patterns of expression. Thus, the complex structure of the dystrophin gene, encoding several large dystrophin-like isoforms and smaller truncated products with different patterns of expression, existed before the divergence between the protostomes and deuterostomes. The conservation of this gene structure in such distantly related organisms, points to important distinct functions of the multiple products.

Renal duplication with associated complications in adults: CT findings in 26 cases.

AIM: To review the computed tomography (CT) findings in 26 adult patients with complicated renal duplication, and to assess whether the complications were anomaly-related or superimposed by acquired disease. MATERIALS AND METHODS: Fifteen women and 11 men, aged 17-83 years took part in the study. All CT studies were reviewed to define the moieties affected. RESULTS: The duplication was unilateral in 18 cases and bilateral in six, one patient had a single left kidney and the remaining one a horseshoe kidney. In 14 patients the pathology was related only to the anomaly. Upper pole abnormalities were seen in 13 patients (seven related to the anomaly) and lower pole abnormalities in five (all related to duplication). Both systems were affected in eight cases, six of them by pathological processes unrelated to duplication. Hydronephrosis of the affected collecting system was the most common imaging finding. CONCLUSION: Computed tomography is often used to evaluate abdominal conditions in adults and may therefore be the first imaging modality to reveal a duplex kidney complicated by a pathological process. Involvement of only one moiety was frequently related to the duplication, with a predilection for the upper moiety, while involvement of both systems was used unrelated to the duplication. Zissin, R. (2001). Clinical Radiology, 56, 58-63.

The cellular prion protein colocalizes with the dystroglycan complex in the brain.

The function of PrP(C), the cellular prion protein (PrP), is still unknown. Like other glycophosphatidylinositol-anchored proteins, PrP resides on Triton-insoluble, cholesterol-rich membranous microdomains, termed rafts. We have recently shown that the activity and subcellular localization of the neuronal isoform of nitric oxide synthase (nNOS) are impaired in adult PrP(0/0) mice as well as in scrapie-infected mice. In this study, we sought to determine whether PrP and nNOS are part of the same functional complex and, if so, to identify additional components of such a complex. To this aim, we looked for proteins that coimmunoprecipitated with PrP in the presence of detergents either that completely dissociate rafts, to identify stronger interactions, or that preserve the raft structure, to identify weaker interactions. Using this detergent-dependent immunoprecipitation protocol we found that PrP interacts strongly with dystroglycan, a transmembrane protein that is the core of the dystrophin-glycoprotein complex (DGC). Additional results suggest that PrP also interacts with additional members of the DGC, including nNOS. PrP coprecipitated only with established presynaptic proteins, consistent with recent findings suggesting that PrP is a presynaptic protein.

Neural network based quantitative structural property relations (QSPRs) for predicting boiling points of aliphatic hydrocarbons

Quantitative structural property relations (QSPRs) for boiling points of aliphatic hydrocarbons were derived using a back-propagation neural network and a modified Fuzzy ARTMAP architecture. With the back-propagation model, the selected molecular descriptors were capable of distinguishing between diastereomers. The QSPRs were obtained from four valance molecular connectivity indices (1chiv,2chiv,3chiv,4chiv), a second-order Kappa shape index (2kappa), dipole moment, and molecular weight. The inclusion of dipole moment proved to be particularly useful for distinguishing between cis and trans isomers. A back-propagation 7-4-1 architecture predicted boiling points for the test, validation, and overall data sets of alkanes with average absolute errors of 0.37% (1.65 K), 0.42% (1.73 K), and 0.37% (1.54 K), respectively. The error for the test and overall data sets decreased to 0.19% (0.81 K) and 0.31% (1.30 K), respectively, using the modified Fuzzy ARTMAP network. A back-propagation alkene model, with a 7-10-1 architecture, yielded predictions with average absolute errors for the test, validation, and overall data sets of 1.96% (6.79 K), 1.83% (6.45 K), and 1.25% (4.42 K), respectively. Fuzzy ARTMAP reduced the errors for the test and overall data sets to 0.19% (0.73 K) and 0.25% (0.95 K), respectively. The back-propagation composite model for aliphatic hydrocarbons, with a 7-9- architecture, yielded boiling points with average absolute errors for the test, validation, and overall set of 1.74% (6.09 K), 1.25% (4.68 K), and 1.37% (4.85 K), respectively. The error for the test and overall data sets using the Fuzzy ARTMAP composite model decreased to 0.84% (1.15 K) and 0.35% (1.35 K), respectively. Performance of the QSPRs, developed from a simple set of molecular descriptors, displayed accuracy well within the range of expected experimental errors and of better accuracy than other regression analysis and neural network-based boiling points QSPRs previously reported in the literature.

Hepatic infarction in preeclampsia as part of the HELLP syndrome: CT appearance.

We describe the computed tomographic (CT) findings of hepatic infarctions in two preeclamptic pregnant women. These infarcts were part of the HELLP syndrome (hemolysis, elevated liver function tests, and low platelets count). In both cases, CT disclosed features characteristic of multiple nonenhancing, low-attenuation, peripheral lesions with vessels coursing through and a mottled appearance. The recognition of such CT findings in liver disease associated with preeclampsia can establish the correct diagnosis.

Myoblast transplantations lead to the expression of the laminin alpha 2 chain in normal and dystrophic (dy/dy) mouse muscles.

Laminin-2 is part of the basement membrane of the skeletal muscle fibers. The laminin alpha 2 chain is absent or drastically reduced in a subgroup of congenital muscular dystrophy patients, and in the severely affected dystrophic dy/dy mouse. We previously reported that heterogeneous primary mouse muscle cell cultures conferred laminin alpha 2 chain expression in dy/dy mice muscles upon cell transplantation. In the present study we investigated whether pure myoblast cell lines were able to confer laminin alpha 2 chain expression in vivo. We observed that: (1) xeno-transplantation of non-immortalized human myoblast in SCID mouse muscles allows human laminin alpha 2 chain expression; (2) allotransplantation of the permanent G8 mouse myoblast cell line in dy/dy muscles allows the expression of the murine laminin alpha 2 chain; and (3) allo-transplantation of the D7 dystrophic dy/dy cell line allows the formation of new and hybrid muscle fibers in dy/dy muscle in the absence of laminin alpha 2 chain expression. We conclude that normal myoblasts are able to restore the expression of an extracellular skeletal muscle protein and that the absence of laminin-2 does not prevent transplanted muscle cells from participating in the formation of myofibers. Myoblasts are, therefore, attractive tools for further exploration of gene complementation strategies in the animal models of congenital muscular dystrophy.

Targeted inactivation of Dp71, the major non-muscle product of the DMD gene: differential activity of the Dp71 promoter during development.

The dystrophin gene, which is defective in Duchenne muscular dystrophy (DMD), also encodes a number of smaller products controlled by internal promoters. Dp71, which consists of the two C-terminal domains of dystrophin, is the most abundant product of the gene in non-muscle tissues and is the major product in adult brain. To study the possible function of Dp71 and its expression during development, we specifically inactivated the expression of Dp71 by replacing its first and unique exon and a part of the concomitant intron with a beta-galactosidase reporter gene. X-Gal staining of Dp71-null mouse embryos and tissues revealed a very stage- and cell type-specific activity of the Dp71 promoter during development and during differentiation of various tissues, including the nervous system, eyes, limb buds, lungs, blood vessels, vibrissae and hair follicles. High activity of the Dp71 promoter often seemed to be associated with morphogenic events and terminal differentiation. In some tissues the activity greatly increased towards birth.

A sea urchin gene encoding dystrophin-related proteins.

The gene which is defective in Duchenne muscular dystrophy (DMD) is the largest known gene. The product of the gene in muscle, dystrophin, is a 427 kDa protein. The same gene encodes at least six additional products: two non-muscle dystrophin isoforms transcribed from promoters located in the 5'-end region of the gene and four smaller proteins transcribed from internal promoters located further downstream. Several other genes, encoding evolutionarily related proteins, have been identified. These include a structurally very similar gene in vertebrates encoding utrophin (DRP1), which is closely related to dystrophin, and a number of small and simple genes in vertebrates or invertebrates encoding proteins similar to some of the small products of the DMD gene. We have isolated a sea urchin gene showing very strong sequence and structural homology with the DMD and utrophin genes. Sequence and intron/exon structure similarities suggest that this gene is related to a precursor of both the DMD gene and the gene encoding utrophin. The sea urchin gene has the unique complex structure of the DMD gene. There is at least one, and possibly more, product(s) transcribed from internal promoters, as well as a large product of >300 kDa containing at least three of the four major domains of dystrophin. The small product seems to be evolutionarily related to Dp116, one of the small products of the human DMD gene. Partial characterization of this gene helped us to construct an evolutionary tree connecting the vertebrate dystrophin gene family with related genes in invertebrates. The constructed evolutionary tree also implies that the vertebrate small and simple structured gene encoding a Dp71-like protein, called DRP2 , evolved from the dystrophin/utrophin ancestral large and complex gene by a duplication of only a small part of the gene.

[Radiologic appearance of "falling gallstones" during laparoscopic cholecystectomy].

Laparoscopic cholecystectomy is the "gold standard" in treating cholelithiasis. Stones are frequently lost in the peritoneal cavity during the procedure, but "missing stones" have been regarded as insignificant. However, there is accumulating evidence that untreated "lost" stones may cause complications even years after operation. We present a 65-year-old woman who presented with vague complaints, anemia and an elevated ESR. CT scan showed an infiltrating process in extra-abdominal muscles compatible with sarcoma. At operation, 2.5 years after previous laparoscopic cholecystectomy, an abscess was found which contained biliary stones. Because of their small size they were not visible on CT scan. We discuss the possible ways of handling "falling stones."

Reduced levels of dystrophin associated proteins in the brains of mice deficient for Dp71.

Duchenne muscular dystrophy (DMD) is a progressive degenerative lethal muscle disease. A significant proportion of DMD affected children suffer also from mental retardation. The rod shaped protein, dystrophin, which is absent from or defective in the muscle of DMD patients, binds to a number of membrane associated proteins (known collectively as dystrophin associated proteins [DAPs]). The levels of DAPs is greatly reduced in the muscle of DMD patients and mdx mice, which lack dystrophin. In addition to dystrophin isoforms, the DMD gene codes also for several smaller proteins. One of the small proteins, Dp71, is expressed in most or all non-muscle tissues and is the major DMD gene product in the brain. The function of the small DMD gene products is unknown. Here we show that mutant mice which do not express the smaller non-muscle products of the DMD gene have a reduced level of DAPs in their brain. This suggests that Dp71 is important for the formation and/or stabilization of a DAPs complex in brain.

[Optic nerve sheath enlargement and reversal of optic nerve head in pseudotumor cerebri].

Using standard cerebral computerized tomography (CT), we diagnosed pseudotumor cerebri (PTC) and correlated the CT findings with CSF pressure and severity of visual impairment. 13 patients with a clinical diagnosis of PTC were compared with 20 age-matched controls with headache, but without papilledema or other neurologic signs. Cerebral CT consisted of axial sections of the posterior fossa, including the orbits. In all subjects the diameter of the optic nerve sheath, reversal of the optic nerve head, presence of empty sella, and size of the ventricles, cisterns and sulci were evaluated. There were no differences in basal cisterns and ventricles between those with PTC and control subjects. Empty sella was found in 6 of 13 PTC patients, compared with 1 of the 20 controls. Optic nerve sheath diameter in controls ranged from 3.5-5.0 mm (average 4.2 +/- 0.54 mm) but from 4.5-9.0 mm (average 6.8 +/- 1.54 mm) in those with PTC. Reversal of the optic nerve head was seen in 4 cases of PTC but in none of the controls. In PTC patients with opening CSF pressure greater than 270 mm water, the diameter of the optic nerve was wider than 7.5 mm. Thus, in most cases of PTC, bilateral enlarged optic nerves can be measured by standard cerebral CT and intracranial space-occupying lesions can be excluded as well. Moreover, reversal of optic nerve head, and empty sella can frequently be seen on CT in those with PTC.

Exogenous Dp71 restores the levels of dystrophin associated proteins but does not alleviate muscle damage in mdx mice.

Dp71 is a non-muscle product of the Duchenne muscular dystrophy gene. It consists of the cysteine-rich and C-terminal domains of dystrophin. We have generated transgenic mdx mice which do not have dystrophin but express Dp71 in their muscle. In these mice, Dp71 was localized to the plasma membrane and restored normal levels of dystrophin associated proteins (DAPs), indicating that Dp71 is capable of interacting with the DAPs in a similar manner to dystrophin. However, the presence of Dp71 and DAPs in the muscle fibres of mdx mice was not sufficient to alleviate symptoms of muscle degeneration.

Detection of Duchenne muscular dystrophy gene products in amniotic fluid and chorionic villus sampling cells.

We have examined the expression of several Duchenne muscular dystrophy (DMD) gene products in amniotic fluid (AF) and chorionic villus sampling (CVS) cells. Variable amounts of dystrophin could be detected in most CVS and AF samples by immunoprecipitation followed by Western blot analysis. PCR analysis demonstrated the presence of the muscle type dystrophin mRNA in all AF cell cultures. The brain type dystrophin mRNA was also detected in some of these cultures. These DMD gene transcripts are of fetal origin and are produced by most or all clonable AF cells. The results may facilitate the development of a method for prenatal diagnosis of DMD, based on the expression of the gene in AF and CVS cells.

A housekeeping type promoter, located in the 3' region of the Duchenne muscular dystrophy gene, controls the expression of Dp71, a major product of the gene.

The 70.8 kDa protein product of the distal part of the giant Duchenne muscular dystrophy (DMD) gene, Dp71, is expressed in many cell types and tissues. Anchored PCR, primer extension and functional analysis of transfected constructs were used to determine the 5' end of the mRNA and characterize the promoter of this major DMD gene product. The 5' untranslated region (5'UTR) of Dp71 is transcribed from a single exon; the promoter does not contain a TATA box, and has a very high GC content and several potential Sp1 binding sites. It is located more than 2000 kb 3' to the muscle and brain type dystrophin promoters and only 150 kb from the 3' end of the gene, suggesting that in most DMD patients the expression of Dp71 is unaffected.

Dp71, the nonmuscle product of the Duchenne muscular dystrophy gene is associated with the cell membrane.

The 70.8 kDa protein, Dp71, is the major Duchenne muscular dystrophy (DMD) gene product in many nonmuscle tissues including the brain. Dp71 shares most of the C-terminal and cysteine-rich domains with the dystrophins but lacks the entire large rod shaped domain of spectrin-like repeats, and the N-terminal actin-binding domain. The function of Dp71 is unknown. Using subcellular fractionation and immunostaining we show that Dp71 is associated with the plasma membrane. Dp71 is also associated with the plasma membrane in mdx myogenic cells transfected with a vector expressing Dp71.