Loss of ubiquitin binding is a unifying mechanism by which mutations of SQSTM1 cause Paget's disease of bone.

Ubiquitin-associated (UBA) domain mutations of SQSTM1 are an important cause of Paget's disease of bone (PDB), which is a human skeletal disorder characterized by abnormal bone turnover. We previously showed that, when introduced into the full-length SQSTM1 protein, the disease-causing P392L, M404V, G411S, and G425R missense mutations and the E396X truncating mutation (representative of all of the SQSTM1 truncating mutations) cause a generalized loss of monoubiquitin binding and impaired K48-linked polyubiquitin binding at physiological temperature. Here, we show that the remaining three known PDB missense mutations, P387L, S399P, and M404T, have similar deleterious effects on monoubiquitin binding and K48-linked polyubiquitin binding by SQSTM1. The P387L mutation affects an apparently unstructured region at the N terminus of the UBA domain, some five residues from the start of the first helix, which is dispensable for polyubiquitin binding by the isolated UBA domain. Our findings support the proposal that the disease mechanism in PDB with SQSTM1 mutations involves a common loss of ubiquitin binding function of SQSTM1 and implicate a sequence extrinsic to the compact globular region of the UBA domain as a critical determinant of ubiquitin recognition by the full-length SQSTM1 protein.

Cloning and Expression of Chinese Duck Interferon-gamma Gene.

The efficacy of cytokine therapy has been demonstrated in several viral diseases. Interferon-gamma is a cytokine that has potent antiviral property and immunomodulatory activity. To investigate the role of IFN-gamma in viral clearance during natural infection and to define the antiviral mechanism, DHBV-infected ducks was used as an animal model. To clone, express, and develop the method of quantifying DuIFN-gamma gene transcription and expression, DuIFN -gamma cDNA was amplified by RT-PCR from PHA stimulated duck PBMC. Recombinant plasmid expressing DuIFN-gamma was used to transfect COS-7, and the cell culture supernatant was analyzed by CPE inhibitory assay and MTT methods to determine the antiviral titer of IFN-gamma. The GST-DuIFN-gamma fusion protein was expressed in E.coli and purified using the GST sepharose 4B. Results indicated that the supernatant collected from COS-7 cells transfected with DuIFN-gamma cDNA was able to prevent duck fibroblasts from VSV induced CPE in a dose dependent manner. An anti-DuIFN-gamma antibody neutralized this antiviral activity.

Exercise-induced hyperkalemia and concentration of Na,K-pumps in skeletal muscle in mitral stenosis: effect of balloon mitral valvotomy.

BACKGROUND AND AIMS OF THE STUDY: The study aim was to examine the effects of balloon mitral valvotomy (BMV) on exercise-induced hyperkalemia, and on changes in the concentration of Na,K-pumps in skeletal muscle, as an exaggerated exercise-induced rise in potassium concentration ([K+]) may contribute to exertional fatigue and breathlessness. METHODS: Eight subjects were evaluated with mitral stenosis (mean age 34 +/- 5.2 years) before, and at two weeks and four months after BMV. Subjects underwent incremental exercise to exhaustion for exercise-induced rise in [K+] and vastus lateralis biopsy for concentration of Na,K-pumps. RESULTS: Mean (+/- SE) valve area increased from 0.89 +/- 0.03 cm2 before to 1.75 +/- 0.05 cm2 after BMV. There was a progressive increase in VO2,max (15.3 +/- 1.6, 17.2 +/- 1.4 and 19.9 +/- 1.9 l/kg/min) at baseline, early after and later after BMV, respectively (p < 0.01). The rise in [K+] with absolute workload fell progressively at early and late follow up post-BMV (p < 0.05), but was unchanged when plotted against percentage of VO2,max to match for relative workload. The concentration of Na,K-pumps was similar to baseline at early follow up (233 +/- 10 versus 228 +/- 15 pmol/g wet weight), but was significantly increased at late follow up after four months (265 +/- 17 pmol/g; p < 0.05). When the relationship between the concentration of Na,K-pumps and the exercise-induced rise in [K+] was studied, a negative correlation was found. However, correlation analysis for the effects of changes in Na,K-pumps on changes in exercise hyperkalemia after BMV was not significant. CONCLUSIONS: The progressive reduction in exercise-induced rise in [K+] after BMV may contribute to the progressive improvement in exercise performance. The increased concentration of Na,K-pumps in skeletal muscle may assist in this improvement, and emphasizes the importance of peripheral adaptations in clinical improvement after BMV.

Dlx5 regulates regional development of the branchial arches and sensory capsules.

We report the generation and analysis of mice homozygous for a targeted deletion of the Dlx5 homeobox gene. Dlx5 mutant mice have multiple defects in craniofacial structures, including their ears, noses, mandibles and calvaria, and die shortly after birth. A subset (28%) exhibit exencephaly. Ectodermal expression of Dlx5 is required for the development of olfactory and otic placode-derived epithelia and surrounding capsules. The nasal capsules are hypoplastic (e.g. lacking turbinates) and, in most cases, the right side is more severely affected than the left. Dorsal otic vesicle derivatives (e. g. semicircular canals and endolymphatic duct) and the surrounding capsule, are more severely affected than ventral (cochlear) structures. Dlx5 is also required in mandibular arch ectomesenchyme, as the proximal mandibular arch skeleton is dysmorphic. Dlx5 may control craniofacial development in part through the regulation of the goosecoid homeobox gene. goosecoid expression is greatly reduced in Dlx5 mutants, and both goosecoid and Dlx5 mutants share a number of similar craniofacial malformations. Dlx5 may perform a general role in skeletal differentiation, as exemplified by hypomineralization within the calvaria. The distinct focal defects within the branchial arches of the Dlx1, Dlx2 and Dlx5 mutants, along with the nested expression of their RNAs, support a model in which these genes have both redundant and unique functions in the regulation of regional patterning of the craniofacial ectomesenchyme.

Magnetic field induced alignment of the directors of a smectic-A liquid crystal.

Deuterium NMR is used to monitor the magnetic field induced alignment of the directors in the smectic A phase of 4-octyl-4(')-cyanobiphenyl. The experiments consist of first preparing the sample as a monodomain by cooling slowly from the nematic phase in the magnetic field of the NMR spectrometer. The tube containing the sample is then rotated quickly through an angle theta(+), and the subsequent field induced alignment process followed by recording deuterium spectra over a period of time. The results obtained show that the magnetic field induced alignment of the directors in a smectic-A sample is qualitatively different from that of nematic samples. Results are reported for the pure mesogen containing deuteriums at the alpha chain position, and these are compared with data obtained on the alignment process by monitoring the deuterium spectrum of a small amount of an added solute, p-xylene-d(10).

Selective sites for polyamine binding to rabbit intestinal brush-border membranes.

The intestinal polyamine transporters have not yet been identified. Our aim was to characterize specific polyamine binding sites in rabbit intestinal brush-border membranes (IBBM) as a starting step for identification of polyamine transporters. This was investigated at 4 degrees and at low membrane concentration. Saturation isotherms for [3H]putrescine (PUT) binding indicated a single population of sites (puT) with a dissociation equilibrium constant Kd of 3.8 microM and a density of sites Bmax of 58 pmol/mg of protein. [3H]spermidine (SPD) binding also involved only one class of sites (spD), albeit with a lower affinity (Kd = 106 microM) and higher abundance (Bmax = 1240 pmol/mg of protein) than puT. On the contrary, [14C]spermine (SPM) bound two classes of sites (spM1 and spM2) differing in their affinity (Kd = 2.5 and 31.4 microM) and abundance (Bmax = 467 and 1617 pmol/mg of protein, respectively). Membrane association of SPM at 4 degrees was much faster than that of SPD and PUT, both of which proceeded at a similar rate. In contrast to PUT and SPD dissociation, SPM dissociation at 23 degrees did not follow a first-order reaction. Specifically bound [3H]PUT, unlike [3H]SPD and [14C]SPM, dissociated at 23 degrees independently of the addition of nonradioactive polyamine. Methylglyoxal-bis-(guanylhydrazone) was an extremely potent inhibitor of PUT binding (Ki = 3.2 +/- 1.5 nM), but as with PUT and cadaverine (CAD), it did not alter [3H]SPD and [14C]SPM binding substantially. The intestinal brush-border membrane may contain at least three sites specific for polyamine binding and exhibiting different ligand selectivity. Site puT might be associated with the transport system already described for intestinal uptake of PUT.

A phase II study of oral clofazimine in unresectable and metastatic hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma is highly refractory to most chemotherapeutic agents. Clofazimine, a riminophenazine compound used to treat leprosy since 1962, inhibits various cancer cell lines, including hepatocellular carcinoma cell lines, via phospholipase A2 dependant processes. Clofazimine also inhibits p170-glycoprotein, the mdr1 gene product. PATIENTS AND METHODS: Thirty patients (26 males and four females) with unresectable (25) or metastatic (5) hepatocellular carcinoma received oral clofazimine 600 mg daily for two weeks, followed by 400 mg daily until progression or death. RESULTS: There were three responses (10%)--one of a soft tissue metastasis, and two of local disease, with 13 patients disease stabilizing for up to 20 months. The overall median survival was 13 weeks. Adverse events included hyperpigmentation, eczematous skin rashes and palpitations. CONCLUSIONS: Although only three patients had an objective response (10%), the 13 patients with stable disease for up to 20 months, and an overall median survival of 13 weeks, suggest that clofazimine, or other riminophenazine compounds may prove to be of value in hepatocellular carcinoma.

Ectopic expression of MITF, a gene for Waardenburg syndrome type 2, converts fibroblasts to cells with melanocyte characteristics.

MITF (microphthalmia-associated transcription factor) encodes a transcription factor with a basic-helix-loop-helix-zipper (bHLH-Zip) motif. MITF mutations occur in patients with Waardenburg syndrome type 2, a disorder associated with melanocyte abnormalities. Here we show that ectopic expression of MITF converts NIH/3T3 fibroblasts into cells with characteristics of melanocytes. MITF transfectants formed foci of morphologically altered cells, which resemble those induced by oncogenes, but did not exhibit malignant phenotypes. Instead, they contained dendritic cells that express melanogenic marker proteins such as tyrosinase and tyrosinase-related protein 1. Most cloned cells of MITF transfectants exhibited dendritic morphology and expressed melanogenic markers, but such properties were not observed in cells transfected with closely related TFE3 cDNA. Our findings indicate that MITF is critically involved in melanocyte differentiation.

Sequence, organization, and transcription of the Dlx-1 and Dlx-2 locus.

There are at least five murine Dlx genes that are related to the Drosophila Distal-less homeobox gene. The Dlx genes are primarily expressed in the developing forebrain, derivatives of the cranial neural crest and restricted epidermal craniofacial and limb domains. Dlx-2 is required for differentiation of subsets of cranial neural crest and forebrain cells. Previous genomic studies have shown that Dlx-1 and Dlx-2 are linked on mouse chromosome 2, near the HoxD cluster. Here we report a detailed analysis of the nucleotide sequence (approximately 14 kb), organization, and transcription of the murine Dlx-1 and Dlx-2 locus. In addition, we show that Dlx-1 makes multiple sense transcripts and at least one antisense transcript, whereas Dlx-2 makes one major transcript. The sequence of the human Dlx-2 gene is reported and is compared to that of the murine gene. Finally, sequence analysis of the deduced protein sequences reveals several candidate functional domains.

Exercise capacity and skeletal muscle structure and function before and after balloon mitral valvuloplasty.

This study evaluated the effects of balloon mitral valvuloplasty (BMV) on exercise capacity and skeletal muscle structure and function in 10 subjects with mitral stenosis (mean age +/- SD 33 +/- 5.5). Measurements were obtained before, and 2 weeks and 4 months after BMV to provide baseline data, to examine the effects of improved hemodynamics, and to examine the effects of resumption of normal physical activity, respectively. Valvuloplasty caused an increase in mitral valve area (0.89 +/- 0.04 to 1.75 +/- 0.07 cm2; mean +/- SE), and an increase in resting cardiac output (3.8 +/- 0.18 to 4.6 +/- 0.19 L/min, p < 0.05). At early follow-up after 2 weeks, subjects did more work (31% increase, p < 0.01) and had greater maximal oxygen consumption (11% increase, p < 0.01). However, measurements reflecting skeletal muscle histology, biochemistry, and function were unaltered at this stage. Four months after BMV, subjects had a further increase in exercise capacity compared with both baseline (58% increase, p < 0.01) and early follow-up (20% increase, p < 0.05). There were associated late increases compared with baseline in quadriceps cross-sectional area (66 +/- 5.8 vs 61 +/- 5.5 cm2, p < 0.05) and torque production (125 +/- 14 vs 118 +/- 16 Nm, p < 0.05). The percentage of slow twitch type I fibers increased compared with baseline (41 +/- 2.0% vs 33 +/- 3.1%, p < 0.05), as did the size of type II fibers (5.9 +/- 0.49 vs 4.9 +/- 0.57 microns2 x 10(3), p < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Expression cloning, developmental expression and chromosomal localization of fibroblast growth factor-8.

A mouse testis cDNA library in lambda pCEV27 eukaryotic expression vector was transfected in NIH3T3 fibroblasts and several transformed foci were identified. A plasmid with high-titered focus forming activity was rescued from one of these transformants. Structural analysis of this cDNA predicted a protein identical to androgen induced growth factor (AIGF), recently identified as the eighth member of the fibroblast growth factor (FGF) family. A 1.6 kilobasepair transcript of the FGF-8 gene was detected in testis but not in other adult tissues analysed. During development, expression of FGF-8 was restricted to embryonic days 9 through 13 suggesting that the growth factor plays a role during a discrete stage of mouse embryogenesis. An exon-containing genomic clone of human FGF-8 was isolated and structural comparisons indicated that the gene structure of this region is highly conserved among the FGF genes. Using a panel of human-rodent somatic cell hybrids, the FGF-8 gene was localized to human chromosome 10.

Oncogene ect2 is related to regulators of small GTP-binding proteins.

We have developed an efficient expression cloning system that allows rapid isolation of complementary DNAs able to induce the transformed phenotype. We searched for molecules expressed in epithelial cells and possessing transforming potential to fibroblasts, and cloned a cDNA for the normal receptor of a growth factor secreted by NIH/3T3 cells. Here we report a second novel transforming gene, ect2. The isolated cDNA is activated by amino-terminal truncation of the normal product. The Ect2 protein has sequence similarity within a central core of 255 amino acids with the products of the breakpoint cluster gene, bcr (ref. 5), the yeast cell cycle gene, CDC24 (ref. 6), and the dbl oncogene. Each of these genes encodes regulatory molecules or effectors for Rho-like small GTP-binding proteins. The baculovirus-expressed Ect2 protein could bind highly specifically to Rho and Rac proteins, whereas the dbl product showed broader binding specificity to Rho family proteins. Thus ect2 is a new member of an expanding family, whose products have transforming properties and interact with Rho-like proteins of the Ras superfamily.

One-year toxicity of orally administered acrolein to the beagle dog.

Forty-eight dogs were separated into four groups of six males and six females. Acrolein (0.1% aqueous) was administered in gelatin capsules to three of these groups at dosing levels of 0.1, 0.5 and 1.5 mg kg-1 day-1 based on results of a range-finding study. After 4 weeks, the high dose was increased to 2 mg kg-1 day-1. The fourth group received deionized water in the same number of gelatin capsules as the high-dose group. Dosing was 7 days per week for 53 weeks. Blood and biochemical measurements were made pretest and at 3-month intervals thereafter. At termination, all dogs were subjected to full necropsy and histological examination. The major test effect noted was frequent vomiting after dosing. This was observed to be dose-dependent and the frequency decreased with time, indicating an adaptive effect. One mid-dose female died during the test and was diagnosed as having died of severe bronchial pneumonia, probably a result of vomitus aspiration. Serum albumin, calcium and total protein values were depressed in high-dose animals throughout the study. Some variability in red blood cell parameters and coagulation times were noted but the significance of these effects was not obvious.

A study of the effect of the irreversible delta receptor antagonist [D-Ala2,Leu5,Cys6]-enkephalin on delta cx and delta ncx opioid binding sites in vitro and in vivo.

Several lines of data support the existence of two classes of delta receptors: the delta cx binding site, which is the delta binding site of the mu-delta opioid receptor complex, and the delta ncx, which is the noncomplexed delta receptor. [D-Ala2,Leu5,Cys6]Enkephalin (DALCE) is an extended analog of [Leu5]enkephalin, which has been shown to bind irreversibly to delta receptors via the terminal cysteine by formation of a disulfide bond with the receptor. In vivo studies have shown that DALCE produces short-lived antinociceptive actions, followed by long-term antagonism of delta receptor-mediated antinociception. The major goal of the present study was to examine the effect of DALCE on the delta cx and delta ncx binding sites in vitro and in vivo. Intracerebroventricular administration of 40 micrograms DALCE failed to decrease [3H][D-Ala2,D-Leu5]enkephalin binding to the delta cx and delta ncx binding sites. Pretreatment of membranes with DALCE in vitro greatly reduced the Bmax of the delta ncx binding site, without significantly altering the Bmax of the delta cx binding site. These findings suggest that when administered in vivo, DALCE fails to distribute uniformly throughout the brain, and that it therefore binds covalently to opioid receptors mostly in the periventricular regions. Viewed collectively, these data support the hypothesis that DALCE acts as a selective delta ncx antagonist, and that the delta ncx binding site, which is sensitive to DALCE, is most likely synonymous with the recently described delta 1 receptor.

Two-year toxicity and carcinogenicity study of acrolein in rats.

Five-hundred and sixty Sprague-Dawley rats were randomized into one control and three treatment groups (70 of each sex per group). Animals were treated by daily gavage with 0.0, 0.05, 0.5 and 2.5 mg kg-1 acrolein in water (10 ml kg-1). These dosing levels were selected as a result of a 6-week range-finding study. Ten rats of each sex per group were sacrificed at 1 year, and the remainder of the animals were treated for 102 weeks. Daily observations wer made, and various clinical, hematological and urine parameters were measured after 3, 6, 12 and 18 months of treatment and immediately prior to termination. All animals, whether found dead or sacrificed, were subject to necropsy and both absolute and relative organ weights were recorded. An extensive array of tissues were examined microscopically for all test animals. The only effects noted for treated rats that were statistically different from controls were consistent depression of creatinine phosphokinase levels, which was difficult to explain, and consistent increases in early cumulative mortalities in both males and females. There was no significantly increased incidence of microscopic lesions in treated rats, whether neoplastic or non-neoplastic. This study clearly demonstrates the lack of neoplastic response in Sprague-Dawley rats as a result of being treated with acrolein by gavage.

Fractal dimensions of cranial sutures and waveforms.

Two quite different shapes of cranial sutures ostensibly yield fractal dimensions. The rare, intricate sutures yield the more valid fractal dimensions because self-similar scaling provides a double-log plot of negative slope. These sutures are fractals over a range of several r values. Some of the highly folded, wavy sutures in humans also fill space except at very tiny values of r, but are nonfractal. A great deal depends on whether the dimension D is > 1 and by how much, whether the curve yields a false fractal dimension, whether the curve scales and shows self-similarity, and whether the scaling occurs regularly in the same pattern. We suggest careful attention to the inverse power law equations, which when misused can yield false fractal values. Cranial sutures vary from the simple wavy sutures to the complex folded ones, and, in rare instances, evolve and develop to the self-similar, scaling, elaborate ones called intricate sutures. The main thing is to express the biology precisely, whether waveform regularity or irregularity or scaling elaboration conserving space and the original shape. D values may not in themselves reliably allow such a distinction, by whatever method used.

Reaction of cytochrome c2 with photosynthetic reaction centers from Rhodopseudomonas viridis.

The reactions of Rhodopseudomonas viridis cytochrome c2 and horse cytochrome c with Rps. viridis photosynthetic reaction centers were studied by using both single- and double-flash excitation. Single-flash excitation of the reaction centers resulted in rapid photooxidation of cytochrome c-556 in the cytochrome subunit of the reaction center. The photooxidized cytochrome c-556 was subsequently reduced by electron transfer from ferrocytochrome c2 present in the solution. The rate constant for this reaction had a hyperbolic dependence on the concentration of cytochrome c2, consistent with the formation of a complex between cytochrome c2 and the reaction center. The dissociation constant of the complex was estimated to be 30 microM, and the rate of electron transfer within the 1:1 complex was 270 s-1. Double-flash experiments revealed that ferricytochrome c2 dissociated from the reaction center with a rate constant of greater than 100 s-1 and allowed another molecule of ferrocytochrome c2 to react. When both cytochrome c-556 and cytochrome c-559 were photooxidized with a double flash, the rate constant for reduction of both components was the same as that observed for cytochrome c-556 alone. The observed rate constant decreased by a factor of 14 as the ionic strength was increased from 5 mM to 1 M, indicating that electrostatic interactions contributed to binding. Molecular modeling studies revealed a possible cytochrome c2 binding site on the cytochrome subunit of the reaction center involving the negatively charged residues Glu-93, Glu-85, Glu-79, and Glu-67 which surround the heme crevice of cytochrome c-554.(ABSTRACT TRUNCATED AT 250 WORDS)