Phenotypes Associated with SHOX Deficiency.

Leri-Weill dyschondrosteosis (LWD) (MIM 127300) is a dominantly inherited skeletal dysplasia characterized phenotypically by Madelung wrist deformity, mesomelia, and short stature. LWD can now be defined genetically by haploinsufficiency of the SHOX (short stature homeobox-containing) gene. We have studied 21 LWD families (43 affected LWD subjects, including 32 females and 11 males, ages 3-56 yr) with confirmed SHOX abnormalities. We investigated the relationship between SHOX mutations, height deficit, and Madelung deformity to determine the contribution of SHOX haploinsufficiency to the LWD and Turner syndrome (TS) phenotypes. Also, we examined the effects of age, gender, and female puberty (estrogen) on the LWD phenotype. SHOX deletions were present in affected individuals from 17 families (81%), and point mutations were detected in 4 families (19%). In the LWD subjects, height deficits ranged from -4.6 to +0.6 SD (mean +/- SD = -2.2 +/- 1.0). There were no statistically significant effects of age, gender, pubertal status, or parental origin of SHOX mutations on height z-score. The height deficit in LWD is approximately two thirds that of TS. Madelung deformity was present in 74% of LWD children and adults and was more frequent and severe in females than males. The prevalence of the Madelung deformity was higher in the LWD vs. a TS population. The prevalence of increased carrying angle, high arched palate, and scoliosis was similar in the two populations. In conclusion, SHOX deletions or mutations accounted for all of our LWD cases. SHOX haploinsufficiency accounts for most, but not all, of the TS height deficit. The LWD phenotype shows some gender- and age-related differences.

The Littler line method and the area under a Gaussian curve: a new method of assessing digital range of motion.

A new method of measuring digital range of motion (the Littler line method) is presented. When a Gaussian curve is centered over the Littler line and the appropriate area under the curve is computed, this area can provide a measure of the functional range of motion regained by an injured digit. Seventeen children (24 digits) with flexor tendon injuries were evaluated at an average follow-up period of 58 months (range, 12-121 months). The Littler line/Gaussian curve method was found to be more reproducible than total active motion, particularly in zone I and II injuries. This method can serve as a more meaningful functional assessment tool than a linear measurement such as total active motion, because it emphasizes digital motion in the mid-ranges of digital motion. (J Hand Surg 2001;26A:23-30.

Treatment of the upper limb in the child with arthrogryposis.

There are good options to maximize upper limb function for the child with arthrogryposis. Preservation of available elbow and wrist motion is essential. A comprehensive plan for upper limb treatment includes operative and nonoperative care.

Madelung's deformity. Surgical correction through the anterior approach.

A resurgence of interest in Madelung's deformity has developed recently because of improved operations for correction of the deformity, identification of the genetic loci for the condition in certain syndromal variants, identification of an anterior ligamentous structure tethering the carpus, and preventive treatments in growing children. The process is reviewed in this article and a new surgical technique is presented. The procedure is performed by way of an anterior incision that is more cosmetically appealing. The release of an anterior ligamentous structure described by Vickers is performed simultaneously with a dome shaped osteotomy of the radius. The fragments, once alignment is corrected, are stablized with temporary pin fixation and a long arm cast until the bone has healed.

Regulation of cell surface expression of CTLA-4 by secretion of CTLA-4-containing lysosomes upon activation of CD4+ T cells.

CTLA-4 is expressed on the surface of activated T cells and negatively regulates T cell activation. Because a low-level expression of CTLA-4 on the cell surface is sufficient to induce negative signals in T cells, the surface expression of CTLA-4 is strictly regulated. We previously demonstrated that the association of CTLA-4 with the clathrin-associated adaptor complex AP-2 induces internalization of CTLA-4 and keeps the surface expression low. However, the mechanism to induce high expression on the cell surface upon stimulation has not yet been clarified. To address this, we investigated the intracellular dynamics of CTLA-4 by analyzing its localization and trafficking in wild-type and mutant CTLA-4-transfected Th1 clones. CTLA-4 is accumulated in intracellular granules, which we identified as lysosomes. CTLA-4 is degraded in lysosomes in a short period, and the degradation process may serve as one of the mechanisms to regulate CTLA-4 expression. Upon TCR stimulation, CTLA-4-containing lysosomes are secreted as proven by the secretion of cathepsin D and beta-hexosaminidase in parallel with the increase of surface expression of CTLA-4 and lysosomal glycoprotein 85, a lysosomal marker. These results suggest that the cell surface expression of CTLA-4 is up-regulated upon stimulation by utilizing a mechanism of secretory lysosomes in CD4(+)T cells.

Tripeptidyl peptidase I, the late infantile neuronal ceroid lipofuscinosis gene product, initiates the lysosomal degradation of subunit c of ATP synthase.

The specific accumulation of a hydrophobic protein, subunit c of ATP synthase, in lysosomes from the cells of patients with the late infantile form of NCL (LINCL) is caused by a defect in the CLN2 gene product, tripeptidyl peptidase I (TPP-I). The data here show that TPP-I is involved in the initial degradation of subunit c in lysosomes and suggest that its absence leads directly to the lysosomal accumulation of subunit c. The inclusion of a specific inhibitor of TPP-I, Ala-Ala-Phe-chloromethylketone (AAF-CMK), in the culture medium of normal fibroblasts induced the lysosomal accumulation of subunit c. In an in vitro incubation experiment the addition of AAF-CMK to mitochondrial-lysosomal fractions from normal cells inhibited the proteolysis of subunit c, but not the b-subunit of ATP synthase. The use of two antibodies that recognize the aminoterminal and the middle portion of subunit c revealed that the subunit underwent aminoterminal proteolysis, when TPP-I, purified from rat spleen, was added to the mitochondrial fractions. The addition of both purified TPP-I and the soluble lysosomal fractions, which contain various proteinases, to the mitochondrial fractions resulted in rapid degradation of the entire molecule of subunit c, whereas the degradation of subunit c was markedly delayed through the specific inhibition of TPP-I in lysosomal extracts by AAF-CMK. The stable subunit c in the mitochondrial-lysosomal fractions from cells of a patient with LINCL was degraded on incubation with purified TPP-I. The presence of TPP-I led to the sequential cleavage of tripeptides from the N-terminus of the peptide corresponding to the amino terminal sequence of subunit c.

Characterization of endopeptidase activity of tripeptidyl peptidase-I/CLN2 protein which is deficient in classical late infantile neuronal ceroid lipofuscinosis.

Endopeptidase activities of the CLN2 gene product (Cln2p)/tripeptidyl peptidase I (TPP-I), purified from rat spleen, were studied using the synthetic fluorogenic substrates. We designed and constructed decapeptides, based on the known sequence cleavage specificities of bacterial pepstatin-insensitive carboxyl proteases (BPICP). MOCAc-Gly-Lys-Pro-Ile-Pro-Phe-Phe-Arg-Leu-Lys(Dnp)r-NH(2) is readily hydrolyzed by Cln2p/TPP-I (K(cat)/K(m) = 7.8 s(-1) mM(-1)). The enzyme had a maximal activity at pH 3.0 for an endopeptidase substrate, but at pH 4.5 with respect to tripeptidyl peptidase activity. Both endopeptidase and tripeptidyl peptidase activities were strongly inhibited by Ala-Ala-Phe-CH(2)Cl, but not inhibited by tyrostatin, an inhibitor of bacterial pepstatin-insensitive carboxyl proteases, pepstatin, or inhibitors of serine proteases. Fibroblasts from classical late infantile neuronal ceroid lipofuscinosis patients have less than 5% of the normal tripeptidyl peptidase activity and pepstatin-insensitive endopeptidase activity. Cln2p/TPP-I is a unique enzyme with both tripeptidyl peptidase and endopeptidase activities for certain substrate specificity.

Trigger finger in children.

At the Texas Scottish Rite Hospital for Children, 239 trigger digits in 176 children were seen and treated surgically over a 10-year period. Trigger fingers accounted for 33 (14%) of these digits in 18 (10%) of the patients. In 8 of 18 patients (44%) the fingers continued to trigger after A-1 pulley release. In children, trigger fingers are different from trigger thumbs. Trigger fingers in children are uncommon and have variable causes, and an A-1 pulley release alone will not always correct the triggering. Additional treatments, such as resection of one or both limbs of the sublimis tendon or an A-3 pulley release, may be required. An awareness of other contributing factors and readiness to explore the entire flexor mechanism should help prevent failure of surgical treatment.

Analysis of where and which types of proteinases participate in lysosomal proteinase processing using bafilomycin A1 and Helicobacter pylori Vac A toxin.

Lysosomal proteinases are translated as preproforms, transported through the Golgi apparatus as proforms, and localized in lysosomes as mature forms. In this study, we analyzed which subclass of proteinases participates in the processing of lysosomal proteinases using Bafilomycin A1, a vacuolar ATPase inhibitor. Bafilomycin A1 raises lysosomal pH resulting in the degradation of lysosomal proteinases such as cathepsins B, D, and L. Twenty-four hours after the withdrawal of Bafilomycin A1, NIH3T3 cells possess these proteinases in amounts and activities similar to those in cells cultured in DMEM and 5% BCS. In the presence of various proteinase inhibitors, procathepsin processing is disturbed by E-64-d, resulting in abnormal processing of cathepsins D and L, but not by APMSF, Pepstatin A, or CA-074. In the presence of Helicobacter pylori Vac A toxin, which prevents vesicular transport from late endosomes to lysosomes, the processing of procathepsins B and D occurs, while that of procathepsin L does not. Thus, procathepsins B and D are converted to their mature forms in late endosomes, while procathepsin L is processed to the mature form after its arrival in lysosomes by some cysteine proteinase other than cathepsin B.

Site-specific localization of Epstein-Barr virus in pharyngeal carcinomas.

In this study, the correlations of factors with Epstein-Barr virus (EBV)-association were investigated in 50 patients with nasopharyngeal carcinoma (NPC), 61 with oropharyngeal carcinoma (OPC), and 55 with hypopharyngeal carcinoma (HPC) in Okinawa and Osaka prefectures in Japan. The incidence of pharyngeal carcinoma in Okinawa was previously found to be higher than that in Osaka; the incidence of OPC was approximately 6 times higher and that of HPC was two times higher. The EBV genome was detected in the tumor cells of the present patients; 83% of the Okinawa and 92% of the Osaka NPC patients. The EBV genome was not detected in OPC or HPC. A univariate analysis showed that sex, the location of the tumor, histology, and the degree of lymphocytic infiltration correlated with the EBV-positive rate. A multivariate analysis revealed that only the location of the tumor was independently correlated with the EBV-positive rate. Histology and tumor size were factors affecting the prognosis of the patients with NPC. The NPC of poorly differentiated type frequently showed the EBV genome, and NPC with lymphocytic infiltration showed a more favorable prognosis compared to the other NPC types. These findings suggest that latent genes of EBV expressed in cancer cells might trigger a cytotoxic T cell reaction against the cancer.

Lipoperoxides in LDL incubated with fibroblasts that overexpress 15-lipoxygenase.

Oxidative modification of LDL plays an important role in early atherogenesis but the mechanisms, nonenzymatic and/or enzymatic, by which LDL is oxidized in vivo remain to be established. Several lines of evidence suggest that cellular 15-lipoxygenase (arachidonate 15-oxidoreductase, EC.1.13.11.13) (15-LO) may contribute to oxidative modification of LDL, including recent studies demonstrating that murine fibroblasts overexpressing 15-LO have an enhanced capacity to oxidize LDL in the medium. The present studies were undertaken to better understand the mechanisms by which cells expressing 15-LO bring about oxidative modification of LDL. LDL incubated 1-2 h with the 15-LO-enriched cells showed a much higher lipoperoxide (LOOH) content than did LDL incubated with control cells. By far the largest absolute increase occurred in cholesteryl ester hydroperoxide (CE-OOH), a much lesser increase in free fatty acid hydroperoxides (FFA-OOH), and only a very small increase in phospholipid hydroperoxides (PL-OOH). Addition of EDTA to the medium abolished these increases in LDL lipid hydroperoxides. Enrichment of LDL with probucol or vitamin E also prevented CE-OOH accumulation. Incubation of LDL with linoleic acid hydroperoxide in the absence of cells also caused a significant increase in CE-OOH and this was markedly inhibited by EDTA. These findings provide further evidence for the potential of 15-LO to participate in LDL oxidation by way of a mechanism involving introduction of LOOH into the LDL particle followed by metal-catalyzed propagation.

Enhanced levels of lipoperoxides in low density lipoprotein incubated with murine fibroblast expressing high levels of human 15-lipoxygenase.

There is strong experimental evidence that oxidized low density lipoprotein (Ox-LDL) plays an important role in atherosclerosis. However, the mechanisms by which Ox-LDL is formed in vivo are unknown. To test whether 15-lipoxygenase (15-LO) could play a role in oxidation of LDL by cells, we expressed 15-LO activity in murine fibroblasts, which do not normally have 15-LO activity, and tested their ability to modify LDL. Using a retroviral vector, we prepared fibroblasts that expressed 2- to 20-fold more 15-LO activity than control fibroblasts infected with a vector containing beta-galactosidase (lacZ). Compared with LDL incubated with lacZ cells, LDL incubated with 15-LO-containing cells were enriched with lipid hydroperoxides. When these LDL samples were subsequently subjected to oxidative stress, they were more susceptible to further oxidative modification, as judged by increased conjugated diene formation and by increased ability to compete with 125I-Ox-LDL for uptake by macrophages. These findings establish that cellular 15-LO can contribute to oxidative modification of LDL, but the quantitative significance of these findings to the in vivo oxidation of LDL remains to be established.

Inhibition of EDRF release by native low-density lipoprotein from cultured porcine endothelial cells through intracellular mechanisms.

Cyclic GMP accumulation in endothelial cells-smooth muscle cells (EC-SMC) coculture induced by both receptor-dependent (thrombin, bradykinin, BK) and receptor-independent (Ca(2+)-ionophore A23187) stimulation, was inhibited by preincubation with low-density lipoprotein (LDL) in time- and concentration-dependent manner. At least 5 min was necessary for the complete blockade with LDL (protein 1 mg/ml). LDL did not affect cyclic GMP-increase by sodium nitroprusside (SNP), a direct stimulator of SMC, but oxidized (ox)LDL (50-250 micrograms/ml) markedly reduced it. An increase of cyclic GMP accumulation in SMC by eluate from stimulated EC columns was completely blocked by 10-min pre-incubation of the column with LDL with or without superoxide dismutase (SOD). In contrast, preincubation of the SMC dish with LDL did not affect cyclic GMP accumulation by the eluate from the stimulated EC column, but preincubation with oxLDL (protein 50-100 micrograms/ml) greatly reduced it. Exposure time of released EDRF to LDL in both coculture and column experiments was < 40-45 s. These results suggest that a brief exposure of EC to pathophysiologic concentration of LDL exclusively affects EC functions, attenuating endothelium-derived relaxing factor (EDRF) release through intracellular mechanisms, and consumption of released EDRF by LDL does not appear to be involved in this LDL inhibitory effect. Possible inhibitory mechanisms of LDL are discussed.

Dynamic external fixation of unstable fractures of the distal part of the radius. A prospective, randomized comparison with static external fixation.

A prospective, randomized study was done to compare the results of dynamic external fixation (the Clyburn device) with those of static external fixation (the AO/ASIF device) in the treatment of fifty unstable fractures of the distal part of the radius. Mobilization of the wrist from neutral to 30 degrees of flexion was begun in the dynamic-fixator group at approximately two weeks, and full motion, allowing 30 degrees of extension, was started at approximately four weeks. The external fixation frames in both groups were kept in place for approximately ten weeks. Mobilization of the wrist in the dynamic-fixator group provided little gain in the mean motion of the wrist at the time of the removal of the fixator or at the one, six, or twelve-month evaluation. The static-fixator group had greater flexion of the wrist and radial deviation at the early and late follow-up examinations, while the dynamic-fixator group demonstrated only greater ulnar deviation one month after the fixator had been removed. Motion of the wrist in the dynamic-fixator group resulted in a statistically significant loss of radial length compared with that in the static-fixator group (four millimeters compared with one millimeter, p < 0.001). Complications were more frequent in the dynamic-fixator group. As evaluated with a modification of the scoring system of Gartland and Werley, 92 percent of the results at one year were excellent or good in the static-fixator group and 76 percent, in the dynamic-fixator group. The results of this study cannot support the concept of early mobilization with a dynamic external fixator for the treatment of unstable fractures of the distal part of the radius.

Enhancement of lysolecithin acyltransferase activity by LDL in thrombin-stimulated porcine-cultured endothelial cells.

Changes of intracellular activity of lysolecithin acyltransferase (LAT) during an interaction between endothelial cells (EC) and low-density lipoprotein (LDL) were investigated. Following an incubation of EC with LDL, endothelial LAT activity was assayed using [3H]lysophosphatidylcholine as the substrate. Stimulation of EC with either thrombin (0.01-1 U/ml) or Ca(2+)-ionophore A23187 (10(-10)-10(-7) M) dose- and time-dependently enhanced LAT activity in the presence of LDL (1 mg protein/ml), but no enhancement was observed in quiescent cells. Ionomycin together with 1-oleoyl-2-acetyl glycerol, a synthetic analog of diacylglycerols enhanced LAT activity in a similar degree to thrombin in the presence of LDL. Either staurosporine, a protein kinase C inhibitor or neomycin, a phospholipase C inhibitor completely blocked an increase of LAT activity in stimulated EC. Stimulation of EC with various agonists including 12-o-tetradecanoylphorbol-13-acetate, an activator of protein kinase C caused a marked increase in cellular uptake of LDL, and staurosporine inhibited the uptake. These results suggest that the transport of LDL into EC is facilitated by stimulation with thrombin and other agonists, and LDL subsequently activates intracellular LAT. Protein kinase C seems to mediate LDL uptake into EC. Intracellular regulatory roles of LDL in the presence of vasoactive substances were discussed.

Isolation and biochemical characterization of procathepsin E from human erythrocyte membranes.

Cathepsin E (CE) is an intracellular, nonlysosomal aspartic proteinase which consists of two identical subunits with a molecular weight of about 42 kDa (K. Yamamoto, M. Takeda, H. Yamamoto, M. Tatsumi, and Y. Kato, 1985, J. Biochem. 97, 821-830). In order to clarify its nature and proteolytic activation, the pro-CE and the mature enzyme were simultaneously purified from human erythrocyte membranes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the reduced proenzyme showed a single protein band, exhibiting an apparent molecular weight of 46 kDa. The proenzyme was found to be catalytically inactive, but it was rapidly converted into the active enzyme after brief acid treatment at pH 4.0, which was accompanied by a reduction in molecular size to 43 kDa. The activated form of pro-CE was essentially identical with the mature enzyme in enzymatic properties such as specific activities toward synthetic and protein substrates, and susceptibilities to various protease inhibitors. The N-terminal amino acid sequence analysis revealed that pro-CE started with the third amino acid residue, Ser3, of the sequence of human gastric CE predicted from its cDNA sequence and that the autocatalytic cleavage occurred at the Met36-Ile37 and Phe39-Thr40 bonds to produce two mature isozymic forms. The mature enzyme purified from human erythrocyte membranes also showed two different N-terminal sequences identical with those of the in vitro-activated form of pro-CE. The proenzyme, as well as the mature enzyme from human and rat erythrocyte membranes, was shown to be as an endo-beta-N-acetylglucosaminidase H-resistant form, whereas CE from rat spleen was N-glycosylated with a high-mannose-type oligosaccharide chain, suggesting that the carbohydrate modification of this protein varies with the cell type or the cellular localization. These data also suggest that the proenzyme in human erythrocytes is processed from the high-mannose type to the complex/hybrid type during its biosynthesis at early stages of erythroid differentiation, which precedes the proteolytic activation.

R1128 substances, novel non-steroidal estrogen-receptor antagonists produced by a Streptomyces. I. Taxonomy, fermentation, isolation and biological properties.

New non-steroidal estrogen-receptor antagonists, R1128 A, B, C and D, were isolated from the cultured broth of Streptomyces sp. No. 1128 by solvent extraction, silica gel chromatography, reverse phase chromatography and preparative HPLC. These compounds inhibited estrogen binding to its receptor. The IC50 values of R1128 A, B, C and D for partially purified rat uterine cytosol receptor were 1.1 x 10(-7) M, 1.2 x 10(-7) M, 2.6 x 10(-7) M and 2.7 x 10(-7) M, respectively.

Procathepsin L-specific antibodies that recognize procathepsin L but not cathepsin L.

Procathepsin L was purified to apparent homogeneity from the culture medium of v-Ha-ras transformed NIH3T3 (Ras-NIH) cells in three steps; anion-exchange chromatography, gel filtration, and re-gel filtration. SDS-PAGE analyses revealed that the purified samples contained only the precursor form, procathepsin L, but not the mature enzyme, cathepsin L. Antibodies against purified procathepsin L were raised. These recognized both rat cathepsin L and the purified procathepsin L. To isolate procathepsin L-specific antibodies that did not recognize cathepsin L, sequential affinity chromatography procedures were carried out. Immunoblot analyses showed that the procathepsin L-specific antibodies recognized only procathepsin L, but not cathepsin L.