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The growth in digital camera usage combined with a worldly abundance of text has translated to a rich new era for a classic problem of pattern recognition, reading. While traditional document processing often faces challenges such as unusual fonts, noise, and unconstrained lexicons, scene text reading amplifies these challenges and introduces new ones such as motion blur, curved layouts, perspective projection, and occlusion among others. Reading scene text is a complex problem involving many details that must be handled effectively for robust, accurate results. In this work, we describe and evaluate a reading system that combines several pieces, using probabilistic methods for coarsely binarizing a given text region, identifying baselines, and jointly performing word and character segmentation during the recognition process. By using scene context to recognize several words together in a line of text, our system gives state-of-the-art performance on three difficult benchmark data sets.

Human adipose tissue secretes a number of proinflammatory mediators that may contribute to the pathophysiology of obesity-related disorders. Understanding the regulatory pathways that control their production is paramount to developing effective therapeutics to treat these diseases. Using primary human adipose-derived stem cells as a source of preadipocytes and in vitro differentiated adipocytes, we found IL-8 and monocyte chemoattractant protein-1 (MCP-1) are constitutively secreted by both cell types and induced in response to serum deprivation. MicroRNA profiling revealed the rapid induction of microRNA 132 (miR-132) in these cells when switched to serum-free medium. Furthermore, miR-132 overexpression was sufficient to induce nuclear factor-kappaB translocation, acetylation of p65, and production of IL-8 and MCP-1. Inhibitors of miR-132 decreased acetylated p65 and partially inhibited the production of IL-8 and MCP-1 induced by serum deprivation. MiR-132 was shown to inhibit silent information regulator 1 (SirT1) expression through a miR-132 binding site in the 3'-untranslated region of SirT1. Thus, in response to nutritional availability, induction of miR-132 decreases SirT1-mediated deacetylation of p65 leading to activation of nuclear factor-kappaB and transcription of IL-8 and MCP-1 in primary human preadipocytes and in vitro differentiated adipocytes.

Interleukin 1beta (IL-1beta) is a potent stimulator of extracellular matrix degradation in models of osteoarthritis (OA). In contrast to bovine explant models which effectively respond to recombinant human IL-1beta, canine models are relatively refractory to human IL-1beta stimulation. Canine IL-1beta cDNA was cloned in order to produce a fully potent species matched preparation of IL-1beta for use specifically in canine models of OA. Established methods for the production of various orthologous IL-1beta proteins from different species are problematic due to the exquisite sensitivity of the mature IL-1beta product to N-terminal variations and the intrinsic technical challenges associated with producing an unmodified product. We have applied a seamless method of SUMO tagging and removal in order to produce a homogeneous unmodified preparation of canine IL-1beta from Escherichia coli which was found to be a potent inducer of aggrecanase activity in isolated canine articular chondrocytes. This method combines highly efficient aspects of seamless plasmid engineering, protein purification, and precise tag removal.

A cDNA was cloned from a rabbit spleen cDNA library which encoded a G-protein alpha subunit peptide of 374 amino acids, that at the peptide level exhibited 86% and 79% identity with human Galpha16 and mouse G(alpha)15, respectively. The rabbit G(alpha)subunit cDNA was subcloned into a mammalian expression vector and transiently co-transfected into HEK-293 cells along with cDNAs encoding the human C3a, C5a, or nociceptin/orphanin FQ receptors. In all three cases the rabbit G alpha subunit behaved similarly to G(alpha)15 or G(alpha)16 and effectively coupled the transfected receptors to intracellular calcium mobilization pathways. By nucleotide sequence homology and functional activity the rabbit G(alpha) subunit appears to be the ortholog of human G(alpha)16 and mouse G(alpha)15.

Cathepsin K is a cysteine protease expressed predominantly in osteoclasts. Activated cathepsin K cleaves key bone matrix proteins and is believed to play an important role in degrading the organic phase of bone during bone resorption. Mutations in the human cathepsin K gene have been demonstrated to be associated with a rare skeletal dysplasia, pycnodysostosis. The degree of functional activity of the mutated forms of cathepsin K in these individuals has not been elucidated, but is predicted to be low or absent. To study the role of cathepsin K in bone resorption, we have generated mice deficient in the cathepsin K gene. Histologic and radiographic analysis of the mice revealed osteopetrosis of the long bones and vertebrae, and abnormal joint morphology. X-ray microcomputerized tomography images allowed quantitation of the increase in bone volume, trabecular thickness, and trabecular number in both the primary spongiosa and the metaphysis of the proximal tibiae. Not all bones were similarly affected. Chondrocyte differentiation was normal. The mice also had abnormalities in hematopoietic compartments, particularly decreased bone marrow cellularity and splenomegaly. The heterozygous animals appeared normal. Close histologic examination of bone histology revealed fully differentiated osteoclasts apposed to small regions of demineralized bone. This strongly suggests that cathepsin K-deficient osteoclasts are capable of demineralizing the extracellular matrix but are unable to adequately remove the demineralized bone. This is entirely consistent with the proposed function of cathepsin K as a matrix-degrading proteinase in bone resorption.

A cDNA clone with 53% amino acid identity to the human type II sodium-dependent phosphate transporter (NaPi-3) was isolated from human small intestine and lung. Functional characterization in Xenopus laevis oocytes showed this cDNA to encode a sodium-dependent phosphate transporter. The electrogenic response is similar to that found in other type II transporters but an inverse pH dependence was observed. By Northern blot, a 4.2-kb transcript was found to be abundantly expressed in lung and, to a lesser degree, in several other tissues of epithelial origin including small intestine, pancreas, prostate, and kidney. This transcript encompasses a 2.073-kb open reading frame which is most closely related (78% amino acid identity) to the mouse sodium-dependent phosphate transporter IIb isoform. This novel transporter, designated human NaPi-3b (Genbank AF111856), appears to be an isoform of the mammalian renal type II co-transporter family., (Copyright 1999 Academic Press.)

Methodology for the production of recombinant active cynomolgus monkey (Macaca fascicularis) cathepsin K (EC 3.4.22.38) was elucidated. The cDNA encoding the cathepsin K was cloned from female M. cynomolgus monkey mRNA. The deduced amino acid sequence of M. cynomolgus preprocathepsin K from the cDNA sequence showed 94.2% identity to human preprocathepsin K. Sequence differences occurred only in the prepro- domains; the mature domains were identical. The recombinant M. cynomolgus cathepsin K was expressed as a secreted proenzyme using baculovirus-infected SF21 insect cells having the predicted N-terminus (LYPEEILDTH ellipsis ), indicating proper cleavage of the secretion sequence. Purified monkey procathepsin K was activated under autocatalytic conditions at pH 4.0. The mature enzyme was composed of mixture of enzymes having N-termini of Gly113 and Arg114. The molecular weight was determined to be 23,668.3 Da by MALDI-TOF-MS which is consistent with the absence of carbohydrate on the mature enzyme. These results indicate that monkey procathepsin K is able to autoactivate and produces a mature enzyme which is identical to that of human cathepsin K. Since the sequence of monkey and human mature cathepsin K are identical and the in vitro activation mechanisms appear to be indistinguishable, monkeys are predicted to be a good animal model for evaluating cathepsin K inhibitors in vivo as therapeutic agents for diseases characterized by excessive bone loss, such as osteoporosis., (Copyright 1998 Academic Press.)

Human cytomegalovirus (CMV) is a member of the Herpesviridae family of viruses that also includes herpes simplex viruses (HSV-1 and HSV-2), varicella-zoster virus (VZV), human herpes virus-6, 7, and 8 (HHV-6, HHV-7, and HHV-8), and Epstein-Barr virus (EBV). Each member of this family encodes a serine protease that is a potential target for antiviral therapeutic intervention. We recently reported the crystal structure of CMV proteases [Qiu, X., Culp, J. S., DiLella, A. G., Hellmig, B., Hoog, S. S., Janson, C. A., Smith, W. W., and Abdel-Meguid, S. S. (1996) Nature 383, 275-279] and proposed that the highly conserved Arg165 and Arg166 residues are involved in stabilizing the oxyanion intermediate in human herpes protease catalyzed reactions through the backbone NH and side chain, respectively. In the current study, site-directed mutagenesis was carried out to probe the catalytic function of these two amino acid residues. Substitution of Arg166 with an alanine has led to ablation of enzymatic activity without detectable change in CMV protease conformation, supporting suggestions from the crystal structure that Arg166 side chain plays a major role in catalysis. The wild-type has a Km = 138 +/- 17 microM and kcat = 19.9 +/- 1.1 min-1, while R166A has only residual activity, with a kcat = 0.012 +/- 0.001 min-1 and an unaltered Km = 145 +/- 18 microM. In the crystal structure, the side chain of Arg166 was shown previously to hold a water molecule that can act as a hydrogen-bond donor to the oxyanion and was thus proposed to stabilize the oxyanion intermediate. However, kinetic characterization of the mutant R165A only reveals a 2.7-fold lower activity than wild-type, with a Km = 166 +/- 19 microM and a kcat = 7.4 +/- 0.4 min-1. These results confirm that Arg165 side chain is not involved in the stabilization of the oxyanion. It is likely that Arg165 only utilizes the backbone NH for catalysis as suggested by the crystal structure.

The prevention of phosphate retention in chronic renal disease may reduce both renal osteodystrophy and disease progression. We evaluated the expression of the sodium-dependent phosphate transporter, NaPi-2, and the response to phosphonoformic acid (PFA) in rats with 5/6 nephrectomy-induced renal failure. Partial nephrectomy resulted in a significant proteinuria and reduced renal function. In addition, there was an approximately 50% reduction in the expression of NaPi-2 mRNA. Treatment of rats for 48 hr with PFA (0.6% in glucose drinking fluid) had no effect on NaPi-2 mRNA; however, PFA resulted in a significant increase in fractional phosphate excretion in both normal (7 +/- 0.5% vs. 3 +/- 0.2%) and uremic (60 +/- 4% vs. 36 +/- 4%) rats. Plasma phosphate concentration was higher in uremic rats (2.5 +/- 0.1 mM) compared with normal rats (1.9 +/- 0.04 mM) but not in uremic rats treated with PFA (2.1 +/- 0.04 mM). These data suggest that PFA can increase renal phosphate excretion independent of changes in phosphate transporter expression and prevent phosphate retention.

A first step in the development of a high-throughput screening assay for antagonists of human E-selectin is the purification and characterization of the selectin. In the present paper we describe a single-step, rapid, reversed-phase HPLC purification protocol for the recombinant, soluble form of human E-selectin (rshE-selectin) produced in Chinese hamster ovary cells. The procedure resulted in high protein yields with recoveries of greater than 98%. Characterization of the reversed-phase purified rshE-selectin showed this product to be analogous to rshE-selectin purified using conventional chromatographic techniques with respect to biological activity and molecular shape. However, the carbohydrate composition of reversed-phase purified rshE-selectin, which had been variable with conventionally purified material, was found to be constant across several isolations. The protocol described herein eliminated the high mannose component associated with previously purified rshE-selectin and provided a uniform carbohydrate composition for additional experimental studies, such as NMR. This fact, coupled with the high yield and simplicity of the present purification scheme are distinct advantages over those previously published. It is expected that other mammalian selectins, such as P-selectin and L-selectin, would also be amenable to reversed-phase HPLC purification.

The gene coding for a soluble form of human E-selectin (sE-selectin) has been expressed in Chinese hamster ovary (CHO) cells. Cells seeded into a hollow fiber reactor secreted protein at a level of 160 mg/liter. The protein was purified to > 95% pure and low endotoxin (< 2 ng/mg), using physiological pH and buffers. The amino acid composition and N-terminal sequence were as predicted from the cDNA sequence. HL-60 cells bound to sE-selectin-coated plates in a dose-dependent manner, and this binding could be blocked up to 100% by pretreatment of HL60 cells with sE-selectin. The concentration of sE-selectin required for 50% inhibition was 1 microM. This value puts an upper limit for the affinity of E-selectin for its natural receptor. sE-selectin also inhibited inflammatory migration of neutrophils in a selective fashion. Purified sE-selectin exhibited a broad band of M(r) approximately 75,000 on nonreducing SDS-PAGE. sE-selectin eluted with M(r) approximately 310,000 from size exclusion chromatography at physiological pH and buffers, suggesting an oligomeric state. Matrix-assisted laser-desorption MS gave a molecular weight of 80,000, while the minimum monomer molecular weight from the gene sequence should be 58,571, demonstrating that the monomeric molecule thus expressed had 27% carbohydrate. Equilibrium analytical ultracentrifugation gave an average solution molecular weight of 81,600 (+/- 4,500). Velocity ultracentrifugation gave a sedimentation coefficient of 4.3 S and, from this, an apparent axial ratio of 10.5:1, assuming a prolate ellipsoid of revolution. An analysis of the NMR NOESY spectra of sE-selectin, sialyl-Lewis X, and sE-selectin with sialyl-Lewis X demonstrates that the recombinant protein binds sialyl-Lewis X productively. Hence, in solution, sE-selectin is a functional elongated monomer.

Site-directed mutants of transforming growth factor-alpha (TGF-alpha) were expressed in an Escherichia coli outer membrane protein A (ompA) expression/secretion vector under the transcriptional control of the lambda PL promoter. TGF-alpha mutant proteins were isolated from cell pellets using alkaline extraction with 0.1 M-Tris (pH 10.5). The levels of protein expression of 23 TGF-alpha mutants were comparable with those of wild-type TGF-alpha, as determined by immunoblotting and radioimmunoassay. An analysis of biological activity using as assays radioreceptor binding competition and colony formation in soft agar showed that the following mutations destroy the activity of TGF-alpha: Gly-19 to Val, Val-33 to Pro and Gly-40 to Val. Mutations of Arg-42 to Lys, Leu-48 to Ala, Tyr-38 to Trp or Phe-17 to Tyr significantly decrease, but do not destroy, biological activity when compared with the wild-type. Mutations in 14 other residues did not significantly alter receptor binding or colony-forming activity. These studies suggest that two domains localized at the surface of TGF-alpha are important in receptor binding and colony-forming activity. Domain I involves amino acid residues which include Tyr-38 and Leu-48; domain II includes residues Phe-15, Phe-17 and Arg-42.

The lumbar shield is a silicone implant designed to prevent postoperative perineural adhesions, indicate a recurrent intervertebral disc rupture, facilitate further surgery at that level, and aid in the postoperative management of the patient. Over three years it has been implanted in 123 experimental patients. There are 83 control patients. Statistical analysis of the results indicates that for the first three months, those patients with the lumbar shield had less postoperative pain. The diagnosis of a recurrent lumbar disc rupture on lateral lumbar spine radiographs is illustrated. The ease of secondary surgery for a recurrent lumbar disc rupture is emphasized. The value of the lumbar shield in postoperative management is discussed, especially in the workmen's compensation/medicolegal groups. One conclusion is that the frequency of multiple secondary surgical procedures on patients with unsatisfactory initial results could be reduced by the use of the lumbar shield.

Postoperative perineural adhesions between the lumbar nerve root and the partially removed intervertebral disc are thought to be a cause of failure of the standard operative procedure for the removal of a ruptured lumbar intervertebral disc. Attempts have been made to reduce postoperative perineural adhesions by the use of epidural muscle, fat, gelatin sponge, silicone, and steroids. The present communication introduces a new implantable silicone device, a lumbar shield, designed to: (a) provide a radiopaque marker on the dorsal perimeter of the excavated lumbar disc so that the presence or absence of a recurrent disc herniation can easily be determined on plain postoperative x-ray films, (b) provide ready access to the operative site in the event of a recurrent disc herniation, (c) prevent postoperative perineural adhesions between the lumbar dura and the nerve root and the partially removed intervertebral disc, and (d) prevent postoperative adhesions between the lumbar dura and the nerve root and the paraspinal muscles. Satisfactory results of lumbar disc surgery over the past 44 years have occurred in about 90% of routine patients. The value of the lumbar shield in 82 patients (59 routine and 23 workmen's compensation/medicolegal patients) followed for 6 months is described. A satisfactory result, i.e., relief of pain or the presence of occasional postoperative pain, occurred in 85% of routine patients at 1 month, 97% at 3 months, and 95% at 6 months.

Tyrosine-specific protein kinases that transfer the terminal phosphate from ATP to protein acceptors are associated with certain transforming viruses and cell surface growth factor receptors. Here we describe the synthesis and testing of potential multisubstrate inhibitors of this class of enzymes. The inhibitors were prepared by covalent attachment of the terminal phosphate of ATP or its tetraphosphate analogue to tyrosine mimics. Testing against p60v-abl, the tyrosine kinase from the Abelson murine leukemia virus, showed that the series of inhibitors was moderately potent (IC50 values as low as 13 microM). However, structural modification of the tyrosine mimic, including replacement with a serine-like moiety, had little effect on potency. It is therefore concluded that the ATP moiety is largely responsible for binding and that the enzyme requires additional structural features for recognition of the tyrosine-containing substrate.

To investigate whether the cell dispersion techniques commonly employed to harvest monolayer cultures of tumor cells for injection into experimental animals might induce alterations in cellular biochemistry, we compared the phosphoprotein profiles of 6 B16 melanoma clones of distinct metastatic potential using 2-D gel electrophoresis after growth in monolayer culture, after suspension by treatment, with trypsin/EDTA and after injection of suspended cells into syngeneic mouse plasma. Trypsin/EDTA treatment and subsequent exposure to syngeneic mouse plasma induced significant alterations in phosphoprotein composition in all clones. Most alterations were quantitative, involving either enhanced or diminished expression of specific phosphoproteins, but qualitative changes involving expression of novel phosphoproteins were also observed. None of the changes in phosphoprotein composition correlated with metastatic potential. The principle alteration induced in all clones by trypsin/EDTA involved enhanced phosphorylation of an NP-40-soluble component with a molecular weight of 79,000 and an isoelectric point of 6.3 [pp79 (6.3)]. This determinant was detected in extracts of B16 monolayer cultures but its level of phosphorylation was enhanced significantly by trypsin/EDTA treatment and by exposure of the harvested cells to syngeneic mouse plasma. These data indicate that procedures commonly employed to harvest tumor cells for assay of tumorigenic and metastatic potential may provide extensive alterations in phosphoprotein composition and that biochemical investigations of tumor cells grown in monolayer culture may not accurately reflect the metabolic status of the same cells immediately prior to and following i.v. injection into experimental animals.

The synthesis and testing of potential multisubstrate inhibitors of tyrosine-specific protein kinases are described. One of the substrates, ATP, was mimicked by the known kinase inhibitor 5'-[4-(fluorosulfonyl)benzoyl]adenosine, which was covalently linked via the sulfonyl moiety to tyrosine mimics. The resulting multisubstrate inhibitors were tested for their ability to inhibit the transfer of phosphate from ATP to a protein acceptor by p60v-abl, the tyrosine kinase encoded by the transforming gene (v-abl) of the Abelson murine leukemia virus (A-MuLV). Although the series of inhibitors displayed moderately potent activity (IC50 values as low as 19 microM), the absence of large effects produced by modification of the tyrosine mimic suggests that they do not behave as multisubstrate inhibitors but bind primarily through the adenosine moiety common to all the inhibitors. This interpretation is strengthened by the finding that the inhibitors lack specificity, inhibiting a serine kinase at comparable concentrations.

The polypeptide and phosphoprotein profiles of a spectrum of B16 melanoma clones of defined metastatic potential have been analyzed by two-dimensional gel electrophoresis. To accommodate the documented instability of metastatic properties in B16 clones, in vitro biochemical assays were always accompanied by in vivo assays of the metastatic behavior using replicate samples of the same clonal populations harvested on the same day. To exclude differences in polypeptide and phosphoprotein profiles resulting from inherent variation in electrophoretic measurements made at different times, polypeptides and phosphoproteins were analyzed in unison for every clone, and a series of clones was examined in parallel in each experiment. Also, samples were electrophoresed simultaneously using a custom-designed apparatus capable of accommodating 20 two-dimensional samples. When tested under these stringent conditions, the polypeptide profiles of B16 clones were indistinguishable. Significant qualitative and quantitative differences in phosphoprotein expression were detected in each clone, but no correlations were found between alterations in protein phosphorylation and metastatic potential. Over 200 discrete phosphoproteins were detected in each clone, but interclonal variation was confined to approximately 10 to 15 phosphoproteins. Expression of three phosphoproteins with the following molecular weights (in kilodaltons) and isoelectric points was strictly qualitative: pp96 (7.9); pp30 (8.2); and pp30 (8.8). In any given clone, they were present individually at equal intensities or were completely absent, but their expression was not coordinate. The data indicate that expression of polypeptide gene products is similar in B16 melanoma clones with widely differing metastatic abilities, but considerable clonal variability exists in posttranslational covalent modification of cell proteins. The possible contribution of protein phosphorylation and other posttranslational pathways in generating the extensive phenotypic heterogeneity observed in tumor cell subpopulations within the same tumor and in the rapid generation of new clonal variants with altered metastatic properties are discussed.

To establish well-characterized cellular reagents for the study of colon carcinoma, we have examined 19 human colorectal carcinoma cell lines with regard to morphology, ultrastructure, expression of tumor-associated antigens, proliferative capacity in vitro, anchorage-independent growth, oncogene expression, tumorigenicity and malignant potential. Cell lines examined were cultured under identical conditions, and in vitro and in vivo analyses were performed in parallel on replicate cultures. Three classes of colorectal cell lines were defined according to their tumorigenicity in nude mice. Class-1 lines formed rapidly progressing tumors in nearly all mice at an inoculum of 10(6) cells. Cell lines belonging to class-2 were less tumorigenic, producing tumors later and at a slower growth rate. Class-3 lines were non-tumorigenic under all experimental conditions tested. By Northern analysis, the oncogenes c-myc, H-ras, K-ras, N-ras, myb, fos and p53 were expressed in nearly all cell lines examined. In contrast, transcripts for abl, src and ros were not detected. The best in vitro predictor of tumorigenicity was colony formation in soft agar. There was no detectable correlation between tumorigenicity and metastatic potential, doubling time in vitro, production of tumor-associated markers, xenograft histology or expression of specific oncogenes.

A thirty-two-year-old male developed multiple neurologic symptoms. Medical evaluation disclosed hypertension, obesity, and a type IV hyperlipoprotein electrophoresis. A nuclear cerebral arteriogram showed reduced flow in the right middle cerebral artery. Arteriographic examination revealed an occlusion at the origin of this artery. The ipsilateral superficial temporal artery was too small to be used in an EC-IC bypass procedure. To correct these conditions, a two-stage procedure was performed. First, an arteriovenous fistula was created between the superficial temporal artery and its accompanying vein. Second, four and one-half months later, the temporal vein, which had by then enlarged, was resected and grafted from the proximal superficial temporal artery to the right angular artery. Angiography three months after the latter procedure showed that the anastomosis was patent.

SEED VIRUS REAGENT AND CONTROL AND IMMUNE ASCITIC FLUIDS WERE PREPARED FOR SIX ARBOVIRUSES: buttonwillow, epizootic hemorrhagic disease of deer, Turlock, anopheles B, Kern Canyon, and Semliki Forest. Pilot studies were initiated for each reagent to determine a satisfactory method for their production. Results of homologous and heterologous tests are reported.