Isolation of a large aggregating proteoglycan from human brain. A large proteoglycan (365 kDa), identified with monoclonal antibodies raised against chondroitin sulfate, was isolated from human brain. The isolation required anion-exchange chromatography followed by gel filtration through a Sephacryl S-500 column. The proteoglycan bound specifically to [3H]hyaluronate (HA). The binding was not reduced by high salt concentrations (up to 4 M) and was inhibited at low pH (< 4.0). The binding was inhibited by the octamer and decamer (but not the hexamer) oligosaccharides of HA. Limited proteolysis of the proteoglycan gave rise to a relatively stable polypeptide (80 kDa). The amino-terminal sequence of the 80-kDa polypeptide was identical to the cDNA-derived amino-terminal sequence of versican, a large human fibroblast proteoglycan. A monoclonal antibody raised against bovine proteoglycans and recognizing the versican core protein reacted by immunoblotting with the proteoglycan isolated from human brain. The antibody was used to localize the proteoglycan in acetone-fixed cryostat sections of bovine spinal cord. The localization of the proteoglycan in the central nervous system was identical to that previously reported for glial hyaluronate-binding protein (GHAP), a 60-kDa glycoprotein of the brain extracellular matrix (ECM). However, a major difference was observed with respect to the sensitivity of the two antigens to hyaluronidase. As previously reported, GHAP was released from the tissue by hyaluronidase digestion, whereas the proteoglycan persisted under these conditions. We conclude that the protein-hyaluronate aggregates in brain ECM contain both GHAP and versican, that GHAP is only retained in the ECM by its interaction with hyaluronate, and that the proteoglycan is anchored in some other manner and probably connects cell surfaces with the ECM since it was not released by hyaluronidase digestion.


Immunoglobulin kappa gene enhancers synergistically activate gene expression but independently determine chromatin structure. Previous studies have located transcriptional enhancer elements within both the intron and 3'-region of the mouse kappa immunoglobulin gene. Here we address the role of these two enhancers in specifying gene activity and specific chromatin structures. MOPC41 kappa gene constructs, either intact or containing deletions of one or both enhancers, were introduced into S194 mouse plasmacytoma cells for transient and stable expression studies. Transient expression assays revealed that the basal level expression exhibited by enhancerless constructs was activated 100-200-fold by the two enhancers together in a synergistic fashion. A similar trend was observed when both enhancers were present in stably integrated constructs, although the synergy was less pronounced. Analysis of DNase I hypersensitive sites in the chromatin revealed that stably integrated constructs established hypersensitive sites about the enhancer sequences. These sites demonstrated the same nuclease susceptibility as those associated with the endogenous gene(s), and their establishment was independent of the presence of the other enhancer. Thus, although both enhancers are required for maximal gene expression, the elements act independently in determining specific chromatin structures.


Identification of basic residues involved in activation and calmodulin binding of rabbit smooth muscle myosin light chain kinase. It is postulated that basic residues in the regulatory region of myosin light chain kinase are important for conferring autoinhibition by binding to the catalytic core. To investigate this proposal, 10 basic amino acids within the regulatory region of rabbit smooth muscle myosin light chain kinase (Lys961-Lys979) were replaced either singularly or in combination with acidic or nonpolar residues by site-directed mutagenesis. All active mutant kinases were dependent on Ca2+/calmodulin for catalytic activity. None of the mutants was active in the absence of Ca2+/calmodulin, suggesting that the autoinhibitory region has not been defined completely. Charge reversal mutants at Arg974, Arg975, and Lys976 resulted in loss of high affinity binding of calmodulin and increased the concentration of calmodulin required for half-maximal activation (KCaM). The charge reversal mutant at Lys979 also increased KCaM but to a lesser extent. Charge reversal mutants at Lys965 and Arg967 resulted in an inactive myosin light chain kinase that could not be proteolytically activated. When these residues were mutated to Ala, the expressed kinase was dependent upon Ca2+/calmodulin for activity and exhibited a decrease in KCaM. Charge reversal mutants in Lys961 and Lys962 also had decreased KCaM values. These basic residues amino-terminal of the calmodulin binding domain may play an important role in the activation of the kinase.


Protein translocation in the endoplasmic reticulum. Ultraviolet light induces the noncovalent association of nascent peptides with translocon proteins. We have photolyzed cell-free translation systems synthesizing beta-lactamase with 254-nm ultraviolet light. In the presence of canine rough microsomes (RM), incomplete chains of beta-lactamase became enriched relative to the full-length molecule in pellet fractions obtained following photolysis and alkaline carbonate extraction. In addition, high molecular weight aggregates were present on SDS-polyacrylamide gels and occurred only when translocation-competent microsomal membranes were used in translation mixtures. The incomplete chains and high molecular weight aggregates were not obtained when RM were inactivated by reaction with N-ethylmaleimide. The incomplete chains did not bind to concanavalin A-Sepharose, indicating that they had not sedimented as a result of being covalently cross-linked to membrane glycoproteins. Both photolysis and alkaline carbonate extraction were required to produce the results. Nascent peptides that were not exposed to alkaline carbonate following photolysis did not appear as high molecular weight bands on SDS-polyacrylamide gels. The high molecular weight aggregates therefore represent denatured protein complexes that contain nascent peptides and microsomal translocon proteins. The results suggest that the translocon is a large proteinaceous complex and that at least a portion of it, when denatured, migrates at a molecular mass of approximately 205 kDa.


The phosphorylation of kinesin regulates its binding to synaptic vesicles. Membrane organella are transported bidirectionally in cells, and the axonal transport system has provided an ideal model system for studying this bidirectional transport. Kinesin and cytoplasmic dynein were identified as candidates for the motor molecules of fast axonal transport, which transport organella along microtubules anterogradely and retrogradely. However, the mechanism that controls this bidirectional transport is unknown. Our previous work revealed that kinesin in axons was associated abundantly with anterogradely transported membranous organella, most of which are believed to be precursors of synaptic vesicles and axonal plasma membranes, while the fractions bound to retrogradely transported ones were very small (Hirokawa, N., Sato-Yoshitake, R., Kobayashi, N., Pfister, K. K., Bloom, G. S., and Brady, S. T. (1991) J. Cell Biol. 114, 295-302). Here we demonstrated in vitro that the binding of kinesin to synaptic vesicles was concentration-dependent and saturable and could be released by high salt concentration. When kinesin was phosphorylated by cAMP-dependent protein kinase, its binding to symaptic vesicles was significantly reduced. By motility assay and by statistical analysis using electron microscopy, we further revealed that synaptic vesicles preincubated with phosphorylated kinesin associated less frequently with microtubules than synaptic vesicles preincubated with unphosphorylated kinesin. The phosphorylation of kinesin should therefore play an essential role in regulating the direction of fast axonal transport by inhibiting its binding to membrane organella, thus releasing it from membrane organella at nerve terminals.


The carboxyl terminus of RAP30 is similar in sequence to region 4 of bacterial sigma factors and is required for function. Transcription factor beta gamma (RAP30/74) from rat liver was previously shown in biochemical studies to control the binding of RNA polymerase II to promoters by a mechanism analogous to that utilized by bacterial sigma factors, by decreasing the affinity of polymerase for nonpromoter sites on DNA and by increasing the affinity of the enzyme for the preinitiation complex (Conaway, R. C., Garrett, K. P., Hanley, J. P., and Conaway, J. W. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 6205-6209). By constructing and analyzing mutants of beta gamma, we have identified a novel functional domain located in the carboxyl terminus of the gamma (RAP30) subunit. This domain shares sequence similarity with region 4 of bacterial sigma factors; in particular, it exhibits striking similarity to the carboxyl-terminal regions 4.1 and 4.2 of SpoIIIC (Bacillus subtilis sigma k). Evidence from biochemical studies argues that a mutant gamma (RAP30), lacking amino acid sequences similar to sigma homology region 4.2, is able to assemble with the beta (RAP74) subunit to form a mutant beta gamma (RAP30/74) with impaired ability to interact with RNA polymerase II.


Alzheimer beta A4-amyloid protein precursor in immunocompetent cells. The mechanism of proteolytic breakdown of the beta A4-amyloid protein precursor (APP) has attracted much attention because of its relevance for Alzheimer's disease. Apart from the pathological role of APP in the amyloidogenesis, many efforts have been made to identify the functional significance of this widely expressed protein in various biological processes. Employing biochemical techniques, we demonstrate that APP is involved in the initiation of the immune response. Upon stimulation, it is expressed by the major functional types of T-lymphocytes, i.e. CD4+ and CD8+ cells. As was demonstrated for the CD4+ lymphoid cell line H9, APP is predominantly secreted. The remaining COOH-terminal fragments generated upon secretion were highly unstable. Of the APP produced by immunocompetent cells, considerable amounts were shown to be leukocyte-derived APP (L-APP). In addition, we were able to identify the KPI-containing L-APP isoform, L-APP733, as the major expressed L-APP isoform in immunocompetent cells, including rat microglial cells and astrocytes. The L-APP expression pattern of these cells showed high similarity. These findings seem to be indicative of an important function of APP within the immune system. Therefore, APP may be involved in various immunopathogenic conditions of the periphery and in the central nervous system.


Interaction between protein kinase C-dependent and G protein-dependent pathways in the regulation of natural killer cell granule exocytosis. The binding of natural killer (NK) cells to either susceptible tumor cells or antibody-coated targets results in rapid activation of phospholipase C (PLC) in NK cells. PLC activation generates inositol-1,4,5-trisphosphate and sn-1,2-diacylglycerol as second messengers, which, in turn, increase intracellular free calcium concentrations ([Ca2+]i) and protein kinase C (PKC) activity, respectively. These proximal signals initiate a cascade of as yet undefined biochemical events, leading eventually to the exocytosis of preformed cytotoxic granules. To investigate the signal transduction pathways involved in granule exocytosis, we utilized streptolysin-O-permeabilized human NK cells as our experimental model. Our initial studies indicated that the separate activation of either PKC (using the phorbol ester, PMA) or G protein-dependent pathways (using guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S)) stimulated granule exocytosis in a time-, concentration-, and Ca(2+)-dependent manner. PMA-stimulated exocytosis was inhibited by staurosporine or a PKC pseudosubstrate antagonist peptide, but was not affected by GDP. In contrast, GTP gamma S-stimulated exocytosis was effectively inhibited by GDP, but not by staurosporine or the PKC pseudosubstrate antagonist. These observations suggest that NK cell exocytosis can be stimulated by at least two separate pathways; one involving PKC and the other involving a G protein. However, co-stimulation with PMA and GTP gamma S synergistically enhanced exocytosis, suggesting that even though the two exocytotic pathways were biochemically distinct, cross-talk between the two pathways may potently influence the exocytotic process. These results define a regulatory role for PKC- and G protein-dependent pathways during granule exocytosis from NK cells.


A key role for a 145-kDa cytosolic protein in the stimulation of Ca(2+)-dependent secretion by protein kinase C. Cytoplasmic Ca2+ is a major regulator of exocytosis in secretory cells; however, the Ca(2+)-dependent mechanisms that trigger secretion have not been elucidated. Protein kinase C (PKC) has been proposed to be an important Ca(2+)-dependent component of this regulation; however, the effects of this enzyme on the exocytotic apparatus have not been identified. We developed a PKC-deficient, semi-intact PC12 cell system in which direct stimulatory effects of purified PKC on Ca(2+)-dependent norepinephrine secretion were studied. The reconstitution of optimal Ca(2+)-activated norepinephrine secretion by semi-intact PC12 cells required the addition of MgATP and cytosolic proteins. PKC-deficient cytosol exhibited reduced reconstituting activity that was fully restored by the addition of purified PKC. The restoration of Ca(2+)-dependent norepinephrine secretion by PKC required the presence of other proteins in the cytosol, in particular, a high molecular weight protein. The high molecular weight protein was identified as p145, a recently characterized 145-kDa brain protein. The addition of PKC enhanced phosphorylation of p145 under conditions of fully reconstituted Ca(2+)-activated norepinephrine secretion. The results indicate that 1) PKC is neither necessary nor sufficient for Ca(2+)-activated secretion, whereas other cytosolic proteins are required; and 2) the stimulation of Ca(2+)-activated secretion by PKC is dependent upon cytosolic proteins such as p145 and may be largely mediated through the phosphorylation of p145.


Vitamin A is a key regulator for cell growth, cytokine production, and differentiation in normal B cells. In the present paper we demonstrate that retinol-retinol-binding protein and chylomicron remnant retinyl esters in concentrations normally found in human plasma inhibit growth of normal human B lymphocytes. Physiological concentrations of retinoic acid (about 30 nM) were less active than physiological concentrations of retinol (about 3 microM). Pharmacological concentrations of retinol and retinoic acid were more active than the concentrations normally found in plasma. Retinol (3 microM) inhibited anti-IgM-mediated DNA synthesis as measured by [3H]thymidine uptake at 72 h by 78%. Furthermore, we found that the cells were blocked in the mid-G1 phase of the cell cycle. Thus, neither MYC up-regulation measured at 3 h nor the expression of the early activation antigen 4F2 was reduced by retinol, whereas the late activation markers (transferrin receptor expression and actinomycin D staining at 48 h of stimulation) were markedly inhibited. Retinol reduced the interleukin 6 production induced by anti-IgM and interleukin 4 after 48 h, whereas the induction of interleukin 6 and tumor necrosis factor by O-tetradecanoylphorbol-13-acetate and ionomycin was less affected. We also noted that the retinoids reduced the formation of plaque-forming cells (i.e. Ig synthesis). These data imply that vitamin A present in human plasma is a normal modulator of B cell function.


Structure and expression of the Kas12 gene encoding a beta-ketoacyl-acyl carrier protein synthase I isozyme from barley. The beta-ketoacyl-acyl carrier protein (ACP) synthase I in the plant fatty acid synthetase catalyzes the condensations of acetate units to a growing acyl-ACP leading to the synthesis of palmitoyl-ACP. Barley chloroplasts contain three cerulenin sensitive beta-ketoacyl-ACP synthase I isoforms, alpha 2, alpha beta, and beta 2. The Kas12 gene encoding the beta 2 isozyme has been isolated and sequenced. The gene spans 3.8 kilobases and contains seven exons separated by six intervening sequences varying from 75 to 1008 base pairs in length. The mosaic gene structure is different compared with that of the beta-ketoacyl synthase in the multifunctional rat and goose fatty acid synthetases. Southern blot analyses of genomic DNA from barley, wheat, and the barley-wheat chromosome addition lines indicate that Kas12 is a single copy gene located on chromosome 2. Primer extension analyses identified four transcription start sites located 168-171 nucleotides upstream from the translation initiation codon. The Kas12 promoter lacks an appropriately positioned TATA box and contains a GC-rich region including two GC elements similar to the Sp1 transcription factor-binding site. In this regard Kas12 closely resembles a set of ubiquitously expressed eucaryotic genes. In accord with this deduction, polymerase chain reaction analysis showed that the Kas12 transcript is present in barley roots, germinating embryos, developing kernels, and leaves.


Electrophoretic studies on the assembly of the nitrogenase molybdenum-iron protein from the Klebsiella pneumoniae nifD and nifK gene products. The electrophoretic properties of the molybdenum-iron (MoFe) protein component of nitrogenase and an iron-molybdenum cofactor (FeMoco)-reactivatable apoMoFe protein from Klebsiella pneumoniae were examined under anaerobic ([O2] < 5 ppm), nondenaturing conditions. In wild type K. pneumoniae extracts, two immunoreactive species migrating more slowly than purified MoFe protein were detected using anti-MoFe protein antibodies. The uppermost species comigrates with the apoMoFe protein produced by a K. pneumoniae mutant unable to synthesize FeMoco (UN106) and by Escherichia coli harboring the plasmids pVL222+pVL15 (nifHDKTYUSWZM+A). In vitro FeMoco titration of the UN106 and pVL222+pVL15 extracts increases the electrophoretic mobility of the apoMoFe protein to that of purified MoFe protein in a two-step process giving rise to a species of intermediate mobility between the apo- and holoMoFe proteins. Two-dimensional gel electrophoresis showed that a 20-kDa peptide is associated with the apoMoFe protein and with the intermediate species, but not with the holoMoFe protein. N-terminal sequencing identified this associated peptide as the nifY gene product, which we propose is acting as a temporary enforcer of the apoMoFe protein structure required for cofactor binding that is released upon FeMoco activation. This FeMoco-induced mobility shift was used to characterize the mutant apoMoFe proteins produced in E. coli as a result of deleting the various nitrogen fixation (nif) genes from the plasmid pVL222. E. coli extracts bearing plasmids deleted in nifH, nifS, nifTYUM, or nifWZM exhibit less than 10% of the apoMoFe protein activity of derepressed UN106 and contain an immunoreactive species whose electrophoretic mobility is increased upon addition of FeMoco from that of apoMoFe protein to that of holoMoFe protein in a single step. Anaerobic nondenaturing gel electrophoresis of 55Fe-labeled E. coli extracts followed by autoradiography showed that these inactive apoMoFe species do not contain iron, indicating that the P-clusters are absent. We therefore propose that NifH, S, U, W, Z, and M are all involved, to varying degrees, in P-cluster assembly. In addition, the presence of the P-clusters does appear to be necessary for the two-step FeMoco activation of the apoMoFe protein to occur.


Pseudomonas exotoxin A-epidermal growth factor (EGF) mutant chimeric protein as an indicator for identifying amino acid residues important in EGF-receptor interaction. Epidermal growth factor (EGF) was fused to the carboxyl end of a modified pseudomonas exotoxin A that has its toxin binding domain deleted. This chimeric toxin designated as PE(delta Ia)-EGF kills A431 cells through the EGF receptor-mediated pathway. In this study, we used a random mutagenesis approach to make point mutations on EGF, followed by replacing the wild type EGF in PE(delta Ia)-EGF with these EGF mutants. We have constructed 14 different PE(delta Ia)-EGFmutants, and examined their EGF receptor binding activity as well as their cytotoxicity to A431 cells. Our results showed that individual mutations of Val19 to Glu and Val34 to Asp in the EGF domain of PE(delta Ia)-EGFmutants resulted in an increase in the binding affinity to EGF receptor and cytotoxicity to A431 cells. On the other hand, individual mutations of His16 to Asp and Gly18 to Ala in the EGF domain of PE(delta Ia)-EGFmutants lead to a decrease in the binding affinity to EGF receptor and cytotoxicity to A431 cells. In addition, mutations of any of the cysteine residues of EGF in PE(delta Ia)-EGFmutants resulted in the loss of their binding activity to EGF receptor and a corresponding loss of their cytotoxicity. This study indicates that the cytotoxicity of PE(delta Ia)-EGFmutant to EGF receptor-bearing cells may be used as an indicator to screen mutations of EGF important in EGF-receptor interactions.


Purification and activation of brain sulfotransferase. Galactosylceramide sulfotransferase (EC 2.8.2.11) catalyzes the biosynthesis of sulfatide from galactocerebroside and adenosine 3'-phosphate 5'-phosphosulfate (PAPS). This enzyme is developmentally controlled, reaching a maximum activity in the brains of mice corresponding to that of maximum myelination. The product, sulfatide, is an important component of myelin. This transferase from mouse brain has been purified 2600-fold using a combination of pyridoxal 5'-phosphate- and ATP-ligated columns. The purified enzyme yielded a single band following SDS-polyacrylamide gel electrophoresis with an apparent M(r) of 31,000. The entire purification procedure can be completed in 1 day. The pH optimum for the enzyme is 7.0. The Km for PAPS is 1.2 x 10(-6) M, and the Km for cerebroside is 2.6 x 10(-5) M. Cerebroside concentrations > 80 pmol/ml are inhibitory. Enzyme preparations were associated with several lipids. Vitamin K+P(i) activated purified preparations of the sulfotransferase and maintained enzyme activity during storage at -80 degrees C.


Structure of the gene encoding dihydrolipoyl transacylase (E2) component of human branched chain alpha-keto acid dehydrogenase complex and characterization of an E2 pseudogene. We have determined the structural organization of the dihydrolipoyl transacylase (E2) gene of the human branched chain alpha-keto acid dehydrogenase complex. The single copy E2 gene spans approximately 68 kilobases of genomic DNA. The complete coding region consisting of the 5'- and 3'-untranslated regions, the mitochondrial targeting sequence (61 amino acids), and the mature E2 sequence (421 amino acids) are encoded by 11 exons ranging from 62 to 2239 base pairs. All the donor and acceptor splice sites conform to the gt-ag rule. Sequence analysis of the promoter-regulatory region showed the presence of a "CAAT box"-like sequence 537 bases upstream of the transcription initiation site. The "TATA box"-like sequence is absent. Also located in this region are sequences resembling glucocorticoid-responsive and cAMP-responsive elements, fat-specific elements, and Sp1- and AP-2-binding sites. Several sets of direct and inverted repeats are also present. Promoter assays using human hepatoma cells (Hep-G2) and Swiss mouse preadipocytes (3T3-L1) showed that a 4.1-kilobase PstI fragment upstream of the transcription start site confers high expression of the luciferase reporter gene. Moreover, an intronless E2 pseudogene was isolated. It corresponds to the complete mitochondrial presequence and the lipoyl-bearing domain that are encoded by exons I through IV of the functional E2 gene. However, the E2 pseudogene contains multiple base changes, deletions, and insertions, and is flanked by short direct repeats. The data indicate that the E2 pseudogene is a retroposon.


Purification of Limulus polyphemus proclotting enzyme. Horseshoe crabs (Limulus polyphemus and Tachypleus tridentatus) possess a proteolytic blood coagulation system within their amebocytes that, after release and endotoxin activation, generates a polymerized insoluble coagulin clot. Clotting enzyme from horseshoe crab amebocyte lysate is the protease that activates the clottable protein (coagulogen) which then forms the coagulin clot. Comparison of the previously published descriptions of this enzyme has revealed significantly discordant biochemical characteristics. We purified a 60-kDa proclotting enzyme from L. polyphemus amebocyte lysate to a single band on polyacrylamide gel electrophoresis. After electrophoresis, evaluation of enzymatic activity of this protein within gels demonstrated that the band of purified protein corresponded to enzymatic activity, as detected by amidolytic activity for chromogenic substrates and by gelation of coagulogen applied to the gel. The enzymatic activity was inhibited by serine protease inhibitors. The purified proclotting enzyme had a molecular weight and amino acid composition different from the previously published characterizations of proclotting enzymes from both L. polyphemus and T. tridentatus.


Deletion of the pro-alpha 1(I) N-propeptide affects secretion of type I collagen in Chinese hamster lung cells but not in Mov-13 mouse cells. We have introduced two mutations into a full-length human pro-alpha 1(I) cDNA that delete 114 amino acids or the entire 139 amino acids of the N-propeptide domain. Wild-type and mutated versions of the cDNA were introduced into cultured Chinese hamster lung (CHL) cells, which do not produce endogenous type I collagen, and into Mov-13 mouse cells, which produce endogenous pro-alpha 2(I) chains but not pro-alpha 1(I) chains. As judged by resistance to proteases, neither mutation impaired intracellular triple helical assembly of human alpha 1(I) homotrimers in CHL cells, or of chimeric type I collagen comprised of human alpha 1(I) and mouse alpha 2(I) chains in Mov-13 cells. Thus, the N-propeptide is not necessary for intracellular assembly of the main helical collagen domain of type I collagen. In CHL cells the rate of secretion of the mutant homotrimers was greatly reduced as compared to wild type homotrimers, and by immunofluorescence and immunoelectron microscopy, the mutant chains were shown to be accumulated in large vesicular expansions of the rough endoplasmic reticulum. When such cells were retransfected with cDNA encoding wild-type human alpha 2(I) chains, mutant alpha 1(I) chains were not rescued and heterotrimers containing the mutant chains were also retained in the intracellular vesicles. By contrast, deletion of the N-propeptide did not affect secretion of heterotrimers containing mutant chains from Mov-13 cells. Thus, an intact N-propeptide appears necessary for efficient secretion of type I collagen from some but not all cell types.


Effects of folate receptor expression following stable transfection into wild type and methotrexate transport-deficient ZR-75-1 human breast cancer cells. Two biochemically distinct systems, the high affinity folate receptor and the lower affinity reduced-folate carrier, have each been implicated in mediating the transport of folates and antifolates into cells. Previous studies from our laboratory have shown that methotrexate accumulation into wild type (WT) ZR-75-1 human breast cancer cells involves a system with characteristics of the reduced-folate carrier, that this system is deficient in methotrexate resistant (MTXR) ZR-75-1 cells in which methotrexate transport is undetectable and that neither breast cancer cell line expresses folate receptors. In this report we examined the possible interaction of the reduced-folate carrier with folate receptors by stably transfecting both WT ZR-75-1 and MTXR ZR-75-1 cells with an expression vector containing a folate receptor cDNA. Clones of stably transfected MTXR ZR-75-1 and WT ZR-75-1 cells expressing comparable levels of folate receptors were studied and compared to the nontransfected cell lines. Although nontransfected WT and MTXR ZR-75-1 cell lines require concentrations > or = 100 nM folic acid for growth, the expression of folate receptors in transfected WT and MTXR ZR-75-1 cells permitted the growth of both cell lines in low concentrations (1 nM) of folic acid. While the defect in the reduced-folate carrier system in MTXR ZR-75-1 cells inhibits their growth in medium containing low concentrations of folinic acid (< or = 1 microM), MTXR ZR-75-1 cells expressing folate receptors display uninhibited growth in 1 nM folinic acid. The accumulation of folic acid, folinic acid, and methotrexate is enhanced in folate receptor-transfected WT ZR-75-1 cells and MTXR ZR-75-1 cells. Furthermore, the accumulation of folates and antifolate was similar in both transfected WT and MTXR ZR-75-1 cell lines that expressed folate receptors. This suggests that alterations in the reduced-folate carrier do not affect folate receptor function. We also examined the effect of folate receptor expression on the sensitivity of WT and MTXR ZR-75-1 cells to methotrexate and to the lipophillic antifolate trimetrexate. Increased folate receptor expression decreased the sensitivity of WT ZR-75-1 cells toward the antifolate trimetrexate, presumably through increased uptake of reduced folates. Although the expression of the folate receptor enhanced the growth of both cell lines in low folate concentrations, it did not affect the sensitivity of either WT or MTXR ZR-75-1 cells to methotrexate.


Agonist action of indole derivatives at 5-HT1-like, 5-HT3, and 5HT4 receptors in vitro. 1. The potency of indole analogues has been studied, in vitro, at 5-hydroxytryptamine4 (5-HT4) receptors mediating contractions of guinea-pig ileum and relaxation of rat oesophagus. These have been compared to other 5-HT receptors in canine saphenous vein (5-HT1-like), rabbit aorta (5-HT2), and guinea-pig ileum (5-HT3). 2. At receptors mediating 5-HT4 responses in ileum and oesophagus, the rank orders of potency were similar. These rank orders differed from those observed at 5-HT1-like, 5-HT2, and 5-HT3 receptors. In particular, 5-hydroxy N,N, dimethyltryptamine but not 5-methoxy N,N, dimethyltryptamine acted as agonists at 5-HT4 receptors. At 5-HT1-like, 5-HT2 and 5-HT3 receptors these compounds were both active. 3. The 5-HT receptors mediating contractions of canine cephalic vein exhibited a rank order profile similar to that observed at receptors mediating contractions of canine saphenous vein, suggesting stimulation of a 5-HT1-like receptor. 4. The rank order of potency of the substituted indoles differed at 5-HT receptors mediating responses in canine saphenous vein, rabbit aorta and guinea-pig ileum (determined in the presence of 5-methoxytryptamine to desensitize 5-HT4 receptors), suggesting the presence of three distinct receptors. Indeed, at 5-HT3 receptors in the ileum, only three agonists (5-HT, 2-methyl-5-HT and 5-hydroxy N,N, dimethyltryptamine) elicited a response, while all remaining compounds were inactive. 5. It is concluded that rank orders of indole potency can prove useful in the delineation of 5-HT subtypes and together with differential antagonist affinities support the existence of four 5-HT receptor subtypes.


Gangliosides prevent the dimethyl sulphoxide-induced increases in [3H]-noradrenaline release from rat isolated atria. 1. In the rat isolated atria labelled with [3H]-noradrenaline ([3H]-NA), the exposure to 1-4% v/v dimethyl sulphoxide (DMSO) during 15 min caused a concentration-dependent increase in the spontaneous outflow of tritiated products, which reached up to 50% with 2% DMSO and up to 100% with 4% DMSO. These effects were entirely prevented by a 2 h in vitro pretreatment with 50 microM bovine brain gangliosides mixture (BBG). 2. The pattern of the spontaneous release of tritiated products was 17.5 +/- 1.9% of [3H]-NA; 38.7 +/- 2.1% of [3H]-3,4-dihydroxyphenylglycol ([3H]-DOPEG); 36.1 +/- 2.4% of [3H]-O-methylated deaminated metabolites ([3H]-OMDA); 4.7 +/- 0.9% of [3H]-3,4-dihydroxymandelic acid ([3H]-DOMA) and 2.9 +/- 0.2% of [3H]-NMN. After 10 min exposure to 2% DMSO, the increase in basal outflow by this agent consisted of 7.4 +/- 2.5% [3H]-NA and 89.0 +/- 3.6% [3H]-DOPEG. The 2 h preincubation with 50 microM BBG protected from the increase of total radioactivity and also from the metabolic alterations caused by DMSO. The BBG per se did not modify either the basal efflux or the metabolic fate of the [3H]-transmitter. 3. In addition to enhancing the spontaneous outflow of radioactivity, the exposure to 2% v/v DMSO increased by 400% the overflow of the [3H]-transmitter elicited by nerve stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)


Contribution of vagal blockade to the tachycardia induced by the antimuscarinic agents atropine and pirenzepine. 1. The cardiac cholinergic blockade and the chronotropic effect of the widely differing antimuscarinic drugs atropine and pirenzepine were investigated in the dog. 2. In conscious dogs, suppression of the parasympathetic system with atropine (0.2 mg kg-1 h-1) causes marked brief cardioacceleration (234 +/- 13 beats min-1) while pirenzepine (3 mg kg-1 h-1) causes moderate but persistent cardioacceleration (179 +/- 13 beats min-1). After suppression of the influence of the cardiac sympathetic system these cardioaccelerator effects are attenuated, particularly those of pirenzepine. 3. The effects of vagal stimulation are blocked completely and persistently by both agents. 4. When the cardioinhibitory action of the vagus nerve is blocked by pirenzepine the induced tachycardia can be increased by atropine, which causes an additional cardioacceleration (25 +/- 9 beats min-1). 5. These results show that the tachycardia induced by antimuscarinic agents is not only due to vagal blockade. The different mechanisms which may be involved are discussed. The results suggest that pirenzepine can suppress cholinergic influence on the heart more electively than atropine, which induced an 'excess tachycardia'. Also, intrinsic heart rate can be approached more closely when pirenzepine is used to suppress the parasympathetic system, than with atropine.


Influence of the substrate binding characteristics of fibronectin on corneal epithelial cell outgrowth. Student Research Award in the Doctoral Degree Candidate Category, Fourth World Biomaterials Congress (18th annual meeting of the Society for Biomaterials), Berlin, Germany, April 24-28, 1992. The outgrowth of corneal epithelial cells onto a polymeric substrate is expected to be the primary event in the epithelialization of a synthetic corneal graft. Circular corneal buttons (5 mm) were punched from excised rabbit corneas and placed onto bare substrates or substrates preadsorbed with fibronectin (fn), albumin, or binary mixtures of both fn and albumin. Cell outgrowth areas were measured after culturing the buttons for 4 days in serum-free medium. Fibronectin adsorption to the materials was measured from pure and binary solutions with 125I-radiolabeled fibronectin. A parameter thought to be related to the binding strength of fn to polymeric substrates was measured in parallel experiments by partial elution of the adsorbed fn by 3% sodium dodecyl sulfate (SDS). Following pure solution fibronectin adsorption a range of outgrowth areas was measured (from 0.86 +/- 0.03 cm2 for glass to 1.49 +/- 0.03 cm2 for TCPS). On all of the materials tested cell outgrowth areas increased following fn preadsorption and decreased following albumin preadsorption relative to bare surfaces (p less than 0.05). Following preadsorption with binary protein mixtures cell outgrowth areas increased with fibronectin adsorption, however, the outgrowth areas were not determined solely by the concentration of fn adsorbed onto the surfaces. This result suggested that the biological efficiency of the adsorbed fibronectin was substrate-dependent. When the cell outgrowth data were cross-plotted against fn retention following SDS elution, the outgrowth areas were found to increase along with increases in fn retention. Based on these data we suggest that epithelial cell outgrowth may be partially governed by the tightness of binding between the fn molecules and the underlying substrate.


In vitro synergistic, antagonistic, and duration of exposure effects of metal cations on eukaryotic cells. Effects of duration of exposure of single metal cations (Ag+1, Cd+2, Cu+2, Ga+3, Ni+2, and Zn+2) on 3H-thymidine (3H-Tdr) incorporation and 3-[4,5-dimethyl-thiazol-2yl-]-2,5-diphenyl tetrazolium bromide-formazan (MTT-f) production in cultured murine fibroblasts (Balb/c 3T3) were investigated, and the synergistic and antagonistic effects of two metal cations applied simultaneously to the fibroblasts were assessed. The effects of duration of exposure were quantified using TC50 values (concentration of an element required to cause 50% toxicity compared with controls) measured after 24, 48, or 72 h. Using MTT-f production, Cd+2, Cu+2, Ga+3, and Ni+2 showed significantly lower TC50 values with increasing time of exposure, whereas the TC50 values for Ag+1 and Zn+2 remained constant. The TC50 values using 3H-Tdr incorporation exhibited a similar pattern with time of exposure, but the effects were less pronounced. The TC50 values for 3H-Tdr and MTT-f tended to equalize at 72 h. All combinations of cations tested (Ag-Cu, Ag-Zn, Ag-Ni, Cu-Zn, Cu-Ni, Ga-Ni, and Cu-Cd) exhibited synergistic and antagonistic effects as measured by MTT-f production. Synergistic and antagonistic effects were not necessarily mutually exclusive in the same system.


Cell-type-specific adhesion mechanisms mediated by fibronectin adsorbed to chemically derivatized substrata. Plasma fibronectin (pFN) adhesion mechanisms on inert substrata were evaluated for murine fibroblasts (3T3) and human neuroblastoma (Platt) cells using glass coverslips chemically derivatized with a self-assembled monolayer of aliphatic chains terminated with a specific endgroup to interact with adsorbed pFN: [CH3], [SH], [SCOCH3], [NH2], [SO3H], or underivatized glass [SiOH]. All surfaces bound similar amounts of pFN and facilitated attachment of both cell types within narrow ranges. However, spreading/differentiation responses of cells differed considerably among the surfaces. While 3T3 cells spread and developed microfilament stress fibers comparably on all surfaces, inclusion of an RGDS-containing synthetic peptide in the medium revealed variation in resistance to stress fiber formation mediated by an RGDS-recognizing integrin: [NH2] greater than [CH3] much greater than [SiOH],[SH],[SCOCH3]. Different patterns of neurite formation were observed for neuroblastoma cells: [SiOH], [SO3H] greater than [SCOCH3],[SH] much greater than [CH3] greater than [NH2]. Similarity in cell responses to both [CH3] and [NH2] surfaces argues against a pattern dependent upon the hydrophobicity of substrata. When pFN was diluted to a subsaturable concentration with albumin for adsorption, neuroblastoma responses changed significantly from those observed on pFN-saturated surfaces, for both spreading and neurite generation: [NH2],[SO3H] much greater than [SH], [SCOCH3] greater than [SiOH],[CH3]. Responses to the pFN: albumin mixture were markedly improved from responses after sequential adsorptions, demonstrating "optimization" of pFN conformation (not merely binding) by coadsorption of albumin molecules. In most cases, the [NH2] surface yielded responses distinctively different from the other surfaces. Overall, these data suggest many variations in the conformation of pFN molecules adsorbed to specific inert surfaces, as well as variations in the responses of cell surface receptors to conformationally specific pFNs. They also reveal cell-type-specific changes in differentiated cell responses on derivatized substrata, mediated by different classes of cell surface receptors for the two cell types, and provide optimism for regulating FN-dependent adhesion mechanisms in either positive or negative contexts on biomaterial surfaces derivatized with one or more of these chemical end-groups.


Effect of surface processing on the attachment, orientation, and proliferation of human gingival fibroblasts on titanium. The adhesion, orientation, and proliferation of human gingival fibroblasts was studied on electropolished (elpTi), etched (etchTi), and sandblasted (sblTi) titanium surfaces. The texture, chemical state, and composition of the titanium surfaces were analyzed using a surface tracing instrument and electron spectroscopy for chemical analysis. Considerable differences were evident in the surface texture and chemical composition of the differently treated titanium plates. Electropolishing produced the smoothest and cleanest surface. Human gingival fibroblasts attached, spread, and proliferated on all titanium surfaces. However, cells on elpTi exhibited an extremely flat morphology and seemed to form cellular bridges with adjacent cells, whereas the etchTi and sblTi surfaces harbored both round and flat cells with many long processes. Cells on elpTi appeared to grow in thick layers with no specific orientation, whereas on etchTi surfaces they were migrating along the parallel, irregular minor grooves caused by mechanical polishing, and on sblTi surfaces they seemed to grow in clusters. Stress-fiber type actin bundles and vinculin-containing focal adhesions were present in cells spreading on elpTi and etchTi surfaces but not in cells spreading on sblTi surfaces. Cell shape, orientation, and proliferation appear to depend on the texture of the titanium surface and probably also on the properties of the oxide layer and adjacent bulk material. Our findings suggest that smooth or finely grooved titanium surfaces could be optimal in implants adjacent to soft tissues as they support the attachment and growth of human gingival fibroblasts.


Polypropylene small-diameter vascular grafts. Polypropylene's physical properties (e.g., high tensile strength) and relatively inert behavior suggest that fabrication into an arterial substitute may result in an efficacious prosthesis. Grafts were woven from polypropylene yarn into conduits 4 mm I.D. x 50 mm in length. Control grafts were Dacron and ePTFE. Baseline platelet aggregometry on all dogs was performed with 10(-5) M ADP. Aspirin and dipyridamole were given for three days preoperatively and maintained for 2 weeks after surgery. Fifty-four grafts were placed into the aortoiliac position, two different graft materials per dog. The grafts were explanted at intervals of 2 weeks through 16 months; photographed for thrombus-free surface area determinations; and preserved for light, scanning, and transmission electron microscopy. Late (4-16 month) patency was 81% (13/16) for polypropylene, 69% (9/13) for Dacron, and 20% (1/5) for ePTFE. These data include one year patencies of 11/12 (92%) for polypropylene and 7/10 (70%) for Dacron. Late patency for polypropylene grafts was better than for PTFE (p less than 0.05). Platelet aggregation status did not predict graft patency. Light microscopy of 2-week polypropylene explants showed inner capsules composed of myofibroblasts and macrophages, with patchy areas of endothelial cells lining the lumen. By 1 month, a confluent endothelialized surface was seen in all polypropylene explants. Progressive thickening of inner capsules with myofibroblasts and collagen continued through 4 months, reaching a mean thickness of 142 +/- 50 microns (compared to 150 +/- 30 microns for Dacron). These findings suggest potential clinical efficacy for polypropylene as an arterial substitute.


Polyurethanes bearing pendant amino acids: fibrinogen adsorption and coagulant properties. Segmented polyurethanes based on 4,4'-diphenylmethane diisocyanate and polypropylene oxide and chain extended with a sulfonated diamine were derivatized by reaction of sulfonate groups in the polymer with amino acids. The chemical composition of the derivatized polymers was determined by elemental analysis. Tensile stress-strain measurements indicated a slight increase in modulus and elongation with incorporation of amino acids. Water uptake at room temperature showed little change following derivatization, but at 70 degrees C increased significantly. Water contact angles were not influenced by the presence of amino acids, but electron spectroscopy for chemical analysis data showed an increase in hard segment content in the near-surface layers so that bulk and surface compositions were more nearly the same in the amino-acid-containing materials. Fibrinogen adsorption from plasma, shown previously to be high on the sulfonated polyurethanes, was reduced by derivatization, due probably to the decrease in free sulfonate content. Thrombin times of plasma in contact with these materials were essentially the same for the derivatized and underivatized materials.


Theoretical analysis on cell size distribution and kinetics of foreign-body giant cell formation in vivo on polyurethane elastomers. The nature of in vivo leukocyte adhesion and foreign-body giant cell (FBGC) formation on polyurethanes was studied through theoretical and statistical analyses in terms of cell size distribution, density changes, and kinetics of FBGC formation. The results showed that the size distribution of FBGCs followed a "most probable" distribution. During FBGC formation, the densities of FBGCs changed with time. At an early stage, the number of FBGCs increased with time to a maximum at the expense of macrophages. As more FBGCs were formed and less macrophages were present, the fusion of FBGCs among themselves became significant. This, in turn, caused a gradual decrease of FBGC density with time. The rate of FBGC formation was characterized by a rate constant that represented certain characteristics of cell fusion and FBGC formation and the density of initial FBGC-forming macrophages that were a small fraction of leukocytes adhering to the surface. The direct correlations of surface cracking and pitting and adherent FBGCs demonstrated the influence of phagocytic actions of FBGCs on the biostability of implanted polyurethanes. While the cracking was thought to be caused by oxidative degradation facilitated by oxygen ion/radical release of FBGCs, the pitting appeared to result from the Methacrol 2138F aggregates diffusing out of the polymer in an acidic microenvironment under FBGCs, which in turn could be enhanced by the surface degradation and cell phagocytosis. The added Santowhite powder in polyurethane had a significant influence on FBGC formation: It reduced FBGC density and rate of FBGC formation by reducing leukocyte adhesion and the number of macrophages participating in FBGC formation.


Synthesis of antibiotic-loaded hydroxyapatite beads and in vitro drug release testing. Various forms of hydroxyapatite (HAP) materials have been developed for use as bone grafts. Since the risk of local infection is associated with surgery, it seems reasonable to incorporate drugs such as antibiotics into implant materials. We therefore investigated the characteristics of drug incorporation into a spherical porous HAP bead (diameter = 8.48 mm, bulk porosity = 0.439) and its in vitro release behavior. Cefotiam (CTM) was used as a model antibiotic. Because of nonuniform pore distribution in the bead, distribution of CTM was estimated from the amounts of CTM experimentally determined at three different sites: the surface, halfway to the center, and the center of the beads. The results indicated that 90% of the drug was incorporated in the concentric outer 0.387 (radius = 1) section of the bead. CTM was released with a short lag time when the dry HAP bead was placed in water. Incorporation of CTM with egg phosphatidylcholine eliminated initial lag time and decreased the release rate of the drug from the bead. The lipid load was useful in controlling the release of CTM from the beads. In addition, to protect relatively unstable drugs from humidity and avoid contamination of drug-incorporated HAP beads an apparatus was designed with which the beads could be enclosed in vials in vacuo and under aseptic conditions on a bench-scale basis.


Synthesis and nonthrombogenicity of polymer membrane with surface-graft polymers carrying thrombin inhibitor. An acrylamide derivative of a thrombin inhibitor was synthesized and graft polymerized to the surfaces of polymer membranes. The thrombin-inhibitor activity was unaffected by the introduction of an acryloyl group. The surface-graft membrane deactivated thrombin markedly and suppressed adhesion of platelets, resulting in a high nonthrombogenicity. Immersion of polymer membranes blended with the thrombin inhibitor in phosphate-buffered saline for 10 d resulted in the loss of nonthrombogenicity, while the polymer membranes grafted with the thrombin inhibitor derivative maintained the nonthrombogenicity over a long period.


Influence of calcium concentration on the dentin wettability by an adhesive. Plane dentin surfaces were abraded perpendicular to the radicular axis of sound human molars. They were cleaned to reveal the tubules, and the morphological features of each surface were studied microscopically. For a first series of teeth, the Vickers microhardness of the dentin surfaces was measured and the calcium and phosphorus composition was determined by electron microprobe analysis. For a second series, the microhardness and wettability of the surface by the Scotchbond adhesive were compared. Positive correlations were found between the following parameters: degree of minerality, dentin compactness, hardness, and spreading capability of the adhesive.


Relations between in vitro cytotoxicity and crosslinked dermal sheep collagens. Collagen-based biomaterials have found various applications in the biomedical field. However, collagen-based biomaterials may induce cytotoxic effects. This study evaluated possible cytotoxic effects of (crosslinked) dermal sheep collagen (DSC) using a 7-d-methylcellulose cell culture with human skin fibroblasts. Non-crosslinked DSC (NDSC), hexamethylene-diisocyanate-crosslinked DSC (HDSC), and glutaraldehyde-crosslinked DSC (GDSC), their extracts (1 x 10 d to 4 x 10 d extracts), or the corresponding extracted DSC samples were tested. Cell growth was evaluated by cell counting, while cell morphology was assessed by light microscopy and transmission-electron microscopy. Both GDSC and, to a lesser extent, HDSC, induced cytotoxicity, observed as inhibited cell growth and deviant cell morphology. The deviant morphology consisted of extensive accumulations of lipid, reduction in the amount and dilatation of rough endoplasmatic reticulum, increased inclusions of cell remnants, and relatively rounded cell membranes. With HDSC, both primary cytotoxicity, due to extractable products from the material, and secondary cytotoxicity, possibly due to a release of cytotoxic products resulting from enzymatic cell-biomaterial interactions, could be discriminated. With GDSC, however, no clear distinction between primary and secondary cytotoxicity could be made. With NDSC, only primary cytotoxicity, measured as low inhibition of cell proliferation, but without deviant morphology, was observed. These remarkable differences in cytotoxicity are discussed in relation to residual agents and specific crosslinks present in DSCs as a consequence of processing and the crosslinking agents used. The residual agents and the specific crosslinks give rise to differences in direct release of products and in sensitivity to hydrolysis and enzymatic breakdown.


Short-term response of brain tissue to cerebrospinal fluid shunts in vivo and in vitro. The purpose of the studies was to determine how gross physical characteristics of cerebrospinal fluid (CSF) shunts and the cellular proliferative response to shunts contribute to shunt obstruction. Ventricular catheters with round holes, slots, and flanges were implanted into the lateral ventricles of rabbits for 4 weeks. All shunt designs were subject to ingrowth of tissue from the ventricle wall or choroid plexus. There were no qualitative or quantitative differences between normal and hydrocephalic rabbits. Astroglial cells from newborn mice were cultured on shunt catheters for 2 or 4 weeks. The growth of these cells was poor, probably because the cells cannot attach well to the silicone rubber substrate. Contact between the shunt catheter and vascularized brain tissue is the most important factor in the genesis of shunt obstruction.


Hydroxylapatite coating of porous implants improves bone ingrowth and interface attachment strength. The effect of a plasma-sprayed hydroxylapatite (HA) coating on the degree of bone ingrowth and interface shear attachment strength was investigated using a canine femoral transcortical implant model. Cylindrical implants were fabricated by sintering spherical Co-Cr-Mo particles 500-710 microns in diameter; the nominal implant dimensions were 5.95 +/- 0.05 mm diameter by 18 mm in length. One half of each implant was coated with hydroxylapatite, 25-30 microns in thickness, by a plasma-spray technique. Using strict aseptic technique, the implants were placed through both femoral cortices into defects approximately 0.05 mm undersized. After 2, 4, 6, 8, 12, 18, 26, and 52 weeks, the implants were harvested and subjected to mechanical pullout testing and undecalcified histologic evaluation. The application of the HA coating to porous implants enhanced both the amount of bone ingrowth and the interface attachment strength at all time periods. These differences were statistically significant for the percent of bone ingrowth at the 4-, 6-, 12-, 18-, 26-, and 52-week time periods, and interface shear strength values were significantly different at the 6-, 8-, 12-, 18-, and 26-week time periods. The rate of development of interface strength and bone ingrowth was also more rapid for the HA-coated implants. No evidence of any disruption, mechanical failure, or biologic resorption of the HA coating was observed. The results of the present study--demonstrating a beneficial effect of the HA coating at all time periods--are believed to be due to the use of paired comparisons, which allow assessment of subtle differences that might otherwise have been obscured by normal biological variability.


Microhardness measurements of renal calculi: regional differences and effects of microstructure. Microhardnesses of five types of renal calculi: calcium apatite (82.5%)/magnesium ammonium phosphate hydrogen (10%)/calcium oxalate monohydrate (7.5%); calcium apatite (95%)/calcium oxalate monohydrate (5%); magnesium ammonium phosphate hydrogen (90%)/calcium apatite (10%); calcium oxalate monohydrate (85%)/calcium apatite (15%); and cystine (100%) were measured. Using Knoop and Vickers indenters the effects of chemical composition and microstructure on the microhardness measurement were assessed. Calcium oxalate monohydrate, magnesium ammonium phosphate hydrogen, and cystine stones, without apparent structure pattern, showed neither regional nor directional differences in their microhardness. In contrast, calcium apatite stones, with distinctly concentric laminae structure, showed regional variations which were correlated with the chemical composition of stone constituents. Scanning electron microscopy of the indenter impressions were taken to help in interpreting the directional dependence in Knoop hardness measurements with respect to the microstructure of the calculi. Vickers measurements showed the crystalline stones were isotropic within a layer. Combined results of Knoop and Vickers measurements indicate that the anisotropic Knoop hardness readings seen in the laminated regions were structural but not material-based. Implications of the results for the fragmentation of renal calculi in extracorporeal shock wave lithotripsy are discussed.


In vitro corrosion fatigue of 316L cold worked stainless steel. The corrosion resistance of 316L cold worked stainless steel depends upon its thin protective oxide layer; and if this is partially broken down, corrosion resistance depends upon its tendency for repassivation. Since the intended function of stainless-steel implants is to sustain musculoskeletal forces, research toward the stability of the oxide film during dynamic loading in simulated bodylike fluids is warranted. A pilot corrosion fatigue study was, therefore, performed on uniaxial tension fatigue specimens cycled to various maximum stress levels near their yield point while immersed in 37 degrees C isotonic saline solution, and combined with the electrochemical insult of (a) imparting an 800 mV vs. SCE anodic potential for 20 s to stimulate local film breakdown, and then (b) returning to a constant 200 mV vs. SCE anodic potential and maintaining that potential during cyclic loading until the specimens broke in two. During the anodic polarization by continuously monitoring the current it was possible to (a) observe the repassivation and corrosion behavior following stimulation, and (b) detect crack initiation, crack propagation and failure onset. The combined effects of accelerated corrosion and mechanical fatiguing disturbed the repassivation tendency and reduced the crack initiation times and the fatigue lives as compared to air and saline controls. As the maximum cyclic load levels were increased, the fatigue lives were further foreshortened.


Tensile testing of tubular vascular grafts produced by thermal compression fusion of flat collagenous materials. A novel process of fusion bonding for the production of vascular grafts has been investigated. This process, based on the application of pressure and heat in a sophisticated way, has been used for the bonding of the longitudinal edges of flat dermal sheep collagen into tubular forms. The method produces continuous overlapping joints which are expected to overcome the problem of coagulation, as occurs in grafts produced by suturing. The values of the measured shear strength of the bond vary from 750 +/- 30 kPa to 940 +/- 32 kPa, depending on tube diameter and overlap length (n = 5). These values are much higher than calculated values of the stress in thin-walled tubes caused by a high static blood pressure, indicating a high safety factor for the fusion-bonded vascular grafts.


PEO enhancement of platelet deposition, fibrinogen deposition, and complement C3 activation. Whereas it has been commonly thought that adding polyethylene oxide PEO to a surface would diminish the capacity of the surface to cause deposition of platelets and of fibrinogen, and to activate complement C3, we present data showing exactly the opposite. These unexpected results are obtained with low molecular weight (2000) PEO, and are not found with higher molecular weight (20,000) PEO.


Calcification of implanted vascular tissues associated with elastin in an experimental animal model. We have previously studied the process of calcification in bioprosthetic porcine heart valves crosslinked with glutaraldehyde. Observations using light microscopy had indicated that calcification of elastic fibers occurs in implanted heart valves, in addition to calcification associated with collagen fibers. To determine the contribution of elastin to the process of calcification, small pieces of rabbit aorta were cross-linked with 0.2% glutaraldehyde, rinsed in buffer, and implanted subcutaneously in young adult male rats. Cross-linked jugular vein implants were included as controls. After an implantation period of 1 month or longer, we observed many areas of calcification in the aortic media associated with elastin and fewer such areas associated with collagen. The elastin-rich aortic tissues accumulated more calcium than venous tissues. Calcium deposits appeared similar in both allogenic and xenogeneic implants. Calcified areas viewed under the electron microscope included intercellular nonfibrous material. Calcified areas involved predominantly the outer layers of elastic fibers. Calcific deposits included needle-like crystals of hydroxyapatite but often consisted of an amorphous flocculant material surrounded by crystals. The close spatial relationship of hydroxyapatite crystals and elastic membranes seen in this study may be relevant to the initiation of dystrophic calcification in glutaraldehyde cross-linked aortic grafts.


Introducing a selectively biodegradable filament wound arterial prosthesis: a short-term implantation study. This article introduces a new compliant and selectively biodegradable filament wound vascular graft and reports the findings of a short-term implantation study. A basic feature of filament winding is its ability to tailor and better control the mechanical properties of the prosthesis, so that a closer match with the anisotropic properties of native arteries is achieved. The elastomeric vascular grafts comprise poly(ether urethane urea) fibers (Lycra) embedded in a two-component matrix consisting of poly(ether urethane) (Pellethane) and a highly flexible poly(ethylene glycol)/poly(lactic acid) biodegradable segmented copolymer (PELA). Typical tensile modulus values fall in the few megapascals (MPa) range, this being comparable to that of natural arteries. The wound graft exhibits excellent handling and suturability characteristics as well as enhanced burst strength. Furthermore, due to its biodegradable constituent, the prosthesis combines minimal intraoperative blood loss and high healing porosity. The graft displays initially negligible in vitro water permeation, which increases gradually with time. In this short-term study, the prostheses were implanted in the canine carotid, and their biological performance was compared to that of expanded Gore-Tex. The luminal surface of the wound grafts was coated with a thin layer of pseudointima, strongly adhered to the prosthesis surface. Contrasting with the very stiff Gore-Tex grafts, the filament wound prostheses retained their high compliance, being highly pulsatile upon explanation. Histological studies fully corroborated these findings, underscoring the healing properties of these new filament wound vascular prostheses.


Antisera binding onto metals immersed in human plasma in vitro. Titanium (Ti), silicon (Si), silver (Ag), vanadium (V), gold (Au), and chromium (Cr) surfaces were made hydrophilic by radiofrequency plasma treatment. The binding of antifibrinogen (a-fib) and anti-high-molecular-weight kininogen (a-HMWK) after incubation of surfaces in 10% human blood plasma was investigated with ellipsometry. Ti and Si surfaces bound a-HMWK but no detectable amounts of a-fib, whereas the other metals bound both types of antisera. Protein adsorption in a fluid gradient produced under a convex lens indicated that the small deposition of a-fib on Ti and Si surfaces is probably a result of protein displacement. This displacement was not evident at lens positions simulating low plasma concentrations where large amounts of a-fib adsorbed. Surfaces washed sequentially in trichloroethane, acetone, and ethanol produced more hydrophobic surfaces and resulted in slower rates of a-fib displacement. These studies would indicate that some metals form complexes with specific plasma proteins or, alternatively, that different metals may bind equal amounts of plasma proteins which express differing antigenicities toward their surroundings.


Human neutrophil response to short-term exposure to F-75 cobalt-based alloy. The effect of biomaterials on the activation of human neutrophils was studied. Human neutrophils were incubated with F-75 cobalt-based alloy or polystyrene microspheres of a nonphagocytosable size with two times total neutrophil plane surface area. Scanning and transmission electron microscope (SEM, TEM), energy dispersive x-ray microanalysis (EDX), and graphite furnace atomic absorption spectroscopy (GFAAS) were used to analyze changes in cellular morphology and metal content. This report presents evidence that human PMNs display morphological changes related to foreign material challenge, including activation on F-75 bead surfaces, pinocytosis of corrosion products, formation of intracellular vacuoles, degranulation, etc. Moreover, when PMNs were present, the corrosion release rate of F-75 increased as much as three times over cell-free controls.


Prevention of tissue calcification on bioprosthetic heart valve by using epoxy compounds: a study of calcification tests in vitro and in vivo. Calcification is the principal cause of the clinical failures of the bioprosthetic heart valves fabricated from glutaraldehyde pretreated porcine aortic valves or bovine pericardium. In this paper, we compared the calcification on various types of bovine pericardiums pretreated with two hydrophilic epoxy compounds adding GA post-treatment (EP 1 and EP 2), glutaraldehyde (GA)- and nontreated pericardium (Fresh), respectively, by in vitro and in vivo tests. Significant decrease of calcification was found by pretreatment with both epoxy compounds rather than with glutaraldehyde: 0.250 +/- 0.001 (Fresh), 0.276 +/- 0.058 (EP 1), 0.302 +/- 0.071 (EP 2), and 0.478 +/- 0.172 (GA) micrograms (Ca)/mg (dried tissue), respectively, after 20 days dipping in a simulating serum solution in vitro; 115.13 +/- 60.11 (Fresh), 129.84 +/- 51.08 (EP 1), 167.39 +/- 20.81 (EP 2), and 205.19 +/- 16.86 (GA) micrograms/mg, respectively, after 3 months subcutaneous implantation in rabbits. The in vitro method for evaluating calcification designed by us gave the similar order among four samples with that obtained by in vivo test. Because the bovine pericardium pretreated with the epoxy compounds adding GA post-treatment possesses the greater tenacity than that pretreated only with epoxy compounds or GA, meanwhile the calcification is also significantly decreased with this pretreatment, it may be expected that the bovine pericardium with this pretreatment will have the greater anticalcification and durability in dynamic stress.


Effect of child survival on contraceptive use in Bangladesh. A study in Bangladesh showed that couples who lost a child often stopped practising contraception in order to have another child. Logistic regression analysis revealed that contraceptive continuation was related to maternal age, parity, husband's education and the sex of the last child.


Estimating infant mortality rates prospectively in Honduras. In order to determine the validity of infant mortality estimates based on retrospective reporting, the Honduran Ministry of Health carried out a follow-up survey of women interviewed in a 1987 national survey. Women were interviewed approximately 14 months after the baseline survey and were asked about the outcomes of their pregnancies and the survival status of their young children. The overall infant mortality rate calculated from the follow-up survey was lower than that obtained from the baseline survey, due to the particularly low rate among the group of women who were pregnant at the time of the baseline survey. Possible explanations for this low rate are discussed.


A hazard logit model analysis of covariates of childhood mortality in Matlab, Bangladesh. In a prospective study in Matlab, a rural area in Bangladesh, the relationship between a variety of covariates and childhood mortality was examined. Economic status of household, education of mother, sex of the children, health intervention programmes, age of mother, and live birth order of the children were identified as having a statistically significant impact on child survival when the effect of age was controlled. The effects of sex of the children, health programmes, age of mother, and birth order were found to be dependent on the age of the children, but the effect of mother's education was dependent on sex of the children.


Determinants of current contraceptive use among Ghanaian women at the highest risk of pregnancy. This study uses data from the Ghana Demographic and Health Survey (GDHS) of 1988 to examine factors determining the continued low levels of contraceptive use in Ghana. The women currently using efficient contraception are those who have sexual intercourse regularly, who discuss family planning with their partner, whose husbands approve of the use of family planning, and who live in the northern sector of the country. The finding that husband's approval is an important determinant of efficient contraceptive use has significant policy implications for Ghana and other African countries, to motivate both husbands and wives to share fertility control responsibilities.


Some determinants of population variation in cortisol levels in a British urban community. Urinary cortisol excretion rates were determined from three urine samples given over 2 days, a rest day and a working day, by 51 men and 50 women. Each subject also completed a questionnaire relating to life style factors and to perceived levels of stress, busyness and happiness on each day. In men, an association between raised cortisol and high levels of stress was found. In women, high levels of busyness were associated with low cortisol excretion rates. The subjective experiences measured accounted for around 10-20% of cortisol variation in this naturalistic setting.


Secular trends of twinning rate in Sweden. The change in parity and maternal age in Sweden accounts for the decline in the twinning rate from the 19th century to the middle of the 20th century. The later decline in twinning rate cannot be explained by changes in maternal age and parity. Fecundity appears to be higher in mothers of twins than in other mothers.


Socioeconomic factors affecting the longevity of the Japanese population: a study for 1980 and 1985. The effects of urbanisation, low income and rejuvenation of the population on life expectancy at birth and at 20, 40 and 65 years of age for males and females in Japan were examined twice, in 1980 and 1985. For males, urbanisation was the major factor determining life expectancy at birth and at age 20 years, and low income was the key determinant of decreased life expectancy except at 65 years of age. For females high income was the factor significantly decreasing life expectancy at 65 years of age in 1980, and rejuvenation of the population inversely influenced life expectancy except at birth in 1985. Life expectancy for all age groups in 1985 was significantly longer than in 1980 for both males and females.


Geographical distribution of some Danish surnames: reflections of social and natural selection. Geographical differences in the frequencies of eight common surnames in Jutland (Denmark) are analysed using data from telephone directories of 121 exchanges. All the names showed a significant geographical surplus variance, which was divided into trend and patchiness components reflecting the history of the names. The surplus variance of surnames with restricted areas of origin was dominated by a large trend component; for surnames with an originally more even distribution, the patchiness component was dominant. The patterns of distribution were affected by processes which modelled natural selection with linkage disequilibrium. The combined patchiness within the clusters of exchanges gave information about patterns of local migration and level of social integration in the communities. Areas situated in boundary regions show high levels of patchiness.


Prevalence and determinants of caesarean section in Jamaica. The prevalence and determinants of primary caesarean section in Jamaica were estimated from a survey of women aged 14-49 years. Among 2328 women reporting 2395 live hospital births during the period January 1984 to May 1989, the prevalence of caesarean section was 4.1%. Repeat caesarean sections accounted for 1.3% of the hospital births during that period. Of the medical complications studied, prolonged labour and/or cephalopelvic disproportion carried the highest risks of primary caesarean section, followed by breech presentation, maternal diabetes, a high birth-weight baby, maternal hypertension, and a low birth-weight baby. The risk of primary caesarean section increased with maternal age, decreased with parity, was higher for urban than for rural residents, and was higher for births in private versus government hospitals.


Measurement of child mortality in association with a multipurpose birth certificate programme in the Southern Highlands Province of Papua New Guinea. A simple and inexpensive method for monitoring child mortality in association with birth registration was introduced into the Southern Highlands of Papua New Guinea. Eight thousand two hundred and one newborn infants were registered in 1988, approximately 77% of all children born in that year. The risk of death by age 2 was determined from reports given by the mother on the present status of a previously born child at the time of a recent delivery or during clinic registration of the current birth. This was 91 per 1000 for the province and corresponds to a risk of death by age 1 of 77/1000 by extrapolation using standard lifetables. This method was validated by comparison with a continuing demographic surveillance system covering 30,000 people in the western part of the province. The new birth certificate has been an incentive to increase supervised delivery rates and to generate a register that can be used to increase vaccination coverage.


Cognitive and educational attainment in different ethnic groups. The educational attainment of over 2000 children aged 7-15 years from six different ethnic groups was assessed. Children of Pakistani and of Bangladeshi origin tended to obtain the lowest mean scores on all tests, often well below those of West Indian children, who tended to perform as well as Indian children and often no worse than the indigenous majority. There were few signs of any reliable trends over age. Differences were more pronounced on tests of reading and vocabulary than on tests of mathematics but, on tests of non-verbal reasoning, most ethnic minorities also obtained low scores. Many of these differences were associated with differences in social and family circumstances.


Relations between fertility, body shape and menopause in Austrian women. The influence of number and date of pregnancies, births, and spontaneous and induced abortions on body shape, age at menopause and menopausal symptoms have been studied for 110 postmenopausal Viennese women. A significant correlation between fertility and age at menopause was not found, but several menopausal symptoms showed statistically significant correlations with fertility. With a higher number of pregnancies the individual symptoms became more severe.


Compression plating for non-union after failed external fixation of open tibial fractures. Fifty non-unions of the tibia that were present in forty-nine patients after external fixation and immobilization in a cast for a high-energy fracture were subsequently treated by compression plating. Initially, there were forty-six open fractures and four closed fractures with a compartment syndrome. Twenty-two non-unions (44 per cent) had early soft-tissue reconstruction (thirteen rotational and nine free flaps). The duration of external fixation averaged ten weeks, and the mean time from injury to plating was eight months. None of the non-unions were infected at the time of plating. The average preoperative deformity in the sagittal (anterior-posterior) plane was 8 degrees and in the frontal (medial-lateral) plane, 9 degrees; after plate fixation, the residual angulation averaged 3 and 2 degrees. Autogenous bone graft was used in thirty-nine of the fifty non-unions. The patients were followed for an average of twenty-four months. Forty-six (92 per cent) of the non-unions united, in an average of seven months, without further intervention. In four patients (8 per cent), the plate broke, necessitating re-plating in three and external fixation in one (the latter patient had an infected non-union). A deep infection developed in three patients (6 per cent). Ultimately, forty-eight non-unions (96 per cent) healed without evidence of infection. Plate osteosynthesis is an effective method of treatment for patients who have had an open fracture of the tibia that has failed to unite after external fixation and immobilization in a cast.


Indirect reduction and tension-band plating of tibial non-union with deformity. Thirty-three patients who had a maligned non-union of the tibial diaphysis were treated by limited open exposure, indirect reduction with a femoral distractor, tension-band plating, lag-screw fixation, and autogenous bone-grafting. The time from the injury to treatment of the non-union averaged twenty-nine months. Twenty-two of the fractures were originally open and sixteen fractures had had a previous infection before treatment of the non-union. The non-unions were classified as hypertrophic in eight patients, oligotrophic in eighteen, and atrophic in seven. All had severe deformity, or the nature or level of the non-union, or both, precluded intramedullary nailing as a treatment option. All thirty-three non-unions healed at an average of four months; the average length of follow-up was nineteen months. The deformity was corrected, within acceptable limits, in thirty-two of the patients. Full motion of the knee was achieved in twenty-nine patients and of the ankle, in eighteen. Complications included four instances of superficial skin breakdowns, one deep infection, and one fracture of the plate. For non-unions of the tibial diaphysis with deformity that are not amenable to intramedullary nailing, the techniques of limited exposure, indirect reduction, tension-band plating, and bone-grafting can yield excellent anatomical and functional results.


The effect of functional knee-braces on strain on the anterior cruciate ligament in vivo. Functional knee-braces are widely used to protect injured or reconstructed anterior cruciate ligaments, despite the fact that few scientific data support their efficacy. We studied seven functional braces, representative of both the typical custom-fit and off-the-shelf designs. The braces were tested on subjects who had a normal anterior cruciate ligament and were scheduled for arthroscopic meniscectomy or exploration of the knee under local anesthesia. After the operative procedure, a Hall-effect strain-transducer was applied to the anterior cruciate ligament. Under low anterior shear loads, two braces provided some protective strain-shielding effect compared with no brace, but this strain-shielding effect did not occur at the higher anterior shear loads expected during the high-stress activities common to athletic events. The DonJoy, Townsend, C.Ti., and Lenox Hill braces demonstrated a strain-shielding effect on the anterior cruciate ligament with an internal torque of five newton-meters applied to the tibia. None of the braces had any effect on strain on the anterior cruciate ligament during active range of motion of the knee from 10 to 120 degrees or during isometric contraction of the quadriceps. Wearing of a brace did not produce an increase in the value for strain on the anterior cruciate ligament. For the activities that were evaluated in this study, none of the braces produced adverse effects on the anterior cruciate ligament, and there were no significant differences in the strain on the anterior cruciate ligament between the use of a custom-fit or an off-the-shelf brace design. There were no apparent advantages of the more expensive custom-made braces compared with the off-the-shelf designs.


Comparison of reconstruction of the anterior cruciate ligament with and without a Kennedy ligament-augmentation device. A randomized, prospective study. In a randomized, prospective study, sixty-four patients who were operated on for a torn anterior cruciate ligament were divided into two groups: thirty patients (the control group) had repair with the Marshall-MacIntosh technique alone, and thirty-four patients had repair with the same technique but with the addition of a Kennedy ligament-augmentation device. The preoperative characteristics were essentially identical in the two groups. The postoperative management, resumption of athletic activities, and occurrence of complications were also similar. All patients were followed for more than two years. No significant functional or clinical difference between the two groups was found with respect to residual laxity determined either from dynamic radiographs or with the KT-1000 arthrometer. There was no evidence that the addition of the Kennedy ligament-augmentation device gave results that were superior to those obtained with the Marshall-MacIntosh technique alone.


Isokinetic and isometric measurement of strength of external rotation and abduction of the shoulder. The strength of active external rotation and of abduction of the shoulder when the humerus was in the plane of the scapula (30 degrees of horizontal flexion anterior to the coronal plane) was measured isokinetically and isometrically in thirty-nine normal volunteers, who were stratified by age and sex. The angles at which peak torque was produced were similar when tested isokinetically and isometrically; these angles were similar for external rotation (at 60 and 30 degrees of internal rotation) and for abduction (at 30 and 60 degrees of abduction). Isometric peak torque was greater than slow-speed (90 degrees per second) isokinetic peak torque, which in turn was greater than fast-speed (210 degrees per second) isokinetic peak torque. There were highly significant differences in strength, measured isokinetically and isometrically, between younger and older men and between older men and older women. The variability of normal values for torque was similar in each group. Repeat testing demonstrated a high reliability of isokinetic measurements and of isometric measurements at angles within the range of the production of peak torque. Complete testing was performed in four normal volunteers before and after a block of the suprascapular nerve. The supraspinatus and infraspinatus components of the rotator cuff contributed a variable proportion to the total strength of abduction (25 to 50 per cent) and external rotation (50 to 75 per cent) throughout the range of motion. This study demonstrated that both isokinetic and isometric testing in the scapular plane are valid methods for measurement of the strength of external rotation and abduction of the shoulder. The data support standardization of the positions for testing the strength of motions of the shoulder: isometric strength of external rotation should be measured in the scapular plane with the shoulder in 45 degrees of abduction and 45 degrees of internal rotation; isometric strength of abduction, in the scapular plane with the shoulder in 45 degrees of abduction; and isokinetic strength of external rotation and abduction, in the scapular plane at 90 degrees per second.


Operative correction of an unstable total hip arthroplasty. We reviewed the results of reoperation in ninety-five patients who had acute subluxation (ten patients) or dislocation (eighty-five patients) of the hip after conventional cemented total hip-replacement arthroplasty. Postoperatively, fifty-eight patients (61 per cent) had no subsequent dislocation or subluxation. Seven of thirty-seven patients who had had recurrent dislocation had occasional subluxation during follow-up. Of the remaining thirty patients in whom instability persisted after the reoperation, twenty-eight had at least one dislocation, and nine had bothersome subluxation. Ten of these thirty-seven patients had another operation for the persistent instability. The causes of instability were classified as malrotation of the component, disruption of the trochanteric-abduction mechanism, impingement, or multiple and unknown, and appropriate treatment was provided. The component was revised in forty-five patients, revision and advancement of the trochanteric component was done in twenty-five patients, and impinging bone or cement was removed from six patients; a combination of these procedures was done in nineteen patients. Over-all, fifty-eight procedures (61 per cent) were successful (no additional subluxations or dislocations). We concluded that the results of operative treatment for an unstable total hip replacement can be optimized when a precise determination of the cause of the instability is made and appropriate measures are applied.


Prophylaxis with indomethacin for heterotopic ossification after Chiari osteotomy of the pelvis. One hundred patients who had painful dysplasia of the hip were treated with a Chiari osteotomy of the pelvis, and the postoperative radiographs were reviewed for the development of heterotopic ossification. Sixty patients did not receive indomethacin postoperatively, and eighteen (30 per cent) had heterotopic ossification of at least class 1; forty patients received indomethacin for two weeks postoperatively, and only three (8 per cent) of them had heterotopic ossification. This difference was significant (p < 0.025). No class-3 or 4 heterotopic ossification was seen in the patients who were given indomethacin. The prevalence of side-effects was relatively low, and no delayed union of the osteotomy was observed in any patient. We concluded that indomethacin, in the dosage schedule and regimen that we used, provides effective prophylaxis for heterotopic ossification after a Chiari pelvic osteotomy.


One-stage correction of the spastic dislocated hip. Use of pericapsular acetabuloplasty to improve coverage. We performed a combined one-stage approach for the treatment of eighteen spastic subluxated or dislocated hips in eleven children who had cerebral palsy. All patients were between five and thirteen years old and had spastic subluxation or dislocation of the hip and severe acetabular dysplasia. The operation consisted of release of the adductors, psoas, and proximal hamstrings; a femoral-shortening varusderotation osteotomy; and a pericapsular pelvic osteotomy. The pelvic osteotomy was designed to increase superolateral coverage of the femoral head in the elongated acetabulum, which had erosion of the superior and lateral aspects. At the latest follow-up (mean duration, six years and ten months), seventeen of the eighteen hips remained anatomically reduced.


Improved preservation of skeletal muscle in amputated limbs using pulsatile hypothermic perfusion with University of Wisconsin solution. A preliminary study. To determine whether pulsatile hypothermic perfusion with University of Wisconsin preservation solution is superior to topical cooling as a method for the preservation of amputated limbs, six pairs of amputated canine limbs were preserved for twelve to fifteen hours. One limb of each pair was subjected to topical cooling and the other, to pulsatile hypothermic perfusion with University of Wisconsin solution. The bioenergetic status of the limbs was monitored by 31phosphorus magnetic-resonance spectroscopy, and histological evaluation was performed to assess ischemic changes in the preserved tissue. The pH and tissue levels of adenosine triphosphate declined three times more slowly in the limbs that were preserved by pulsatile hypothermic perfusion than in the topically cooled limbs. Consistent with these findings, the perfused limbs also had less histological evidence of ischemic injury. The data from this in vitro study show that pulsatile hypothermic perfusion with University of Wisconsin solution, in combination with an optimum degree of topical cooling, is superior to topical cooling alone as a method of preserving the bioenergetic status of amputated limbs.


Juvenile hallux valgus. A conservative approach to treatment. A conservative method consisting of use of a thermoplastic splint at night as well as passive and active exercises was used for the treatment of hallux valgus in fifty-six children and teen-agers who were between one month and sixteen years old. The splint was remodeled periodically. At the time of follow-up, which ranged from two to six years, the metatarsophalangeal joint angle or the intermetatarsal angle, or both, had improved in approximately half of the feet. No recurrences were detected among the patients who improved.


Changes in growth-plate morphology associated with rejection of rat-limb allografts. Histological and electron microscopic studies were performed to demonstrate the changes in the morphology of the growth plate that occur in allografts obtained from the limbs of growing rats. A genetically defined model was used in which the right hindlimbs of Lewis rats were orthotopically transplanted into Fischer-344 recipient rats. These strains are matched for major histocompatibility antigens but mismatched for minor histocompatibility antigens. The disparity at the minor histocompatibility complex between the Lewis donors and the Fischer recipients creates a weak histocompatibility barrier to transplantation. Lewis-to-Lewis syngeneic limb grafts were used as controls. The proximal parts of the transplanted tibiae were excised during acute rejection of the allograft on days 1, 5, 8, 11, 14, and 28 postoperatively. During rejection, a widened zone of calcified cartilage in the growth plate was observed at eleven days; this zone increased progressively thereafter. The number of chondroclasts in the primary spongiosa of the metaphysis had decreased significantly at eleven days, and chondroclasts had disappeared completely at fourteen days, in association with mononuclear cell infiltration. Electron microscopic examination revealed inactive morphology in some chondroclasts at eight days, and the number of inactive chondroclasts had increased significantly on day 11. At fourteen days, there were no viable chondroclasts in the primary spongiosa, and only remnants of degenerated chondroclasts were present. These findings suggest that the chondroclasts were early targets of rejection and their loss resulted in the cessation of resorption of the calcified cartilage. However, the proliferation and maturation of chondrocytes in the growth plate and the calcification of the matrix continued, despite progression of rejection in the metaphysis. Thus, survival of the chondrocytes and rejection of the chondroclasts apparently led to the formation of a widened calcifying zone in the growth plate.


Distal lengthening of the hamstrings in patients who have cerebral palsy. Long-term retrospective analysis. One hundred and twenty-six patients who had cerebral palsy and who had been managed with distal lengthening of the hamstrings were followed for three to fourteen years so that the long-term results of the procedure could be determined. Twenty-four of sixty-two patients who could not walk before the procedure were able to walk about the house postoperatively. Mild recurvatum developed in only ten patients, and twenty-two patients had a reoperation because of recurrence. Although straight-leg raising and the popliteal angle had improved markedly at one year in all but two patients, both parameters regressed gradually over time. Of several selected variables, only limitation of straight-leg raising preoperatively was of statistical significance (p < 0.001) for the prediction of recurrence. Age, preoperative walking ability, concomitant operations, and postoperative immobilization had no significant effect on the rates of recurrence or recurvatum.


The Ellison iliotibial-band transfer for a torn anterior cruciate ligament of the knee. Long-term follow-up. We studied the long-term results of the Ellison technique of extra-articular transfer of the iliotibial band, without advancement of the biceps tendon, as the sole operative treatment for a torn anterior cruciate ligament. Thirty-two patients (thirty-two knees) were evaluated an average of eleven years (range, seven to fifteen years) after the operation. The average age at the time of the operation was twenty-two years (range, sixteen to thirty-four years). Eighteen patients (56 per cent) had a modified Lysholm score of less than 84 points, indicating symptoms during the activities of daily living. Only six patients (19 per cent) had a subjectively normal knee (a modified Lysholm score of more than 94 points). The most common reason for a poor subjective score was the presence of symptoms of instability, in twenty-four patients (75 per cent). Twenty-four patients (75 per cent) had a positive pivot-shift test and twenty-nine patients (91 per cent), a positive Lachman test. Twelve patients (38 per cent) had severe (grade 3 or 4) radiographic changes. The radiographs of the knee appeared normal (grade 0) in only eight patients (25 per cent). There was a significant association between a meniscal injury and radiographic changes (p < 0.05). Fourteen patients (44 per cent) had subsequent procedures due to persistent instability or pathological changes in the articular cartilage or in a meniscus. There was a significant decline in the subjective and objective knee scores in the twenty-one patients who were evaluated at both two and eleven years. The number of patients who had a positive pivot-shift test increased from five (24 per cent) to sixteen (76 per cent). Subjectively, the number of patients who had a good result decreased from fourteen (67 per cent) to five (24 per cent). Objectively, nine patients (43 per cent) had a rating of good at two years; this fell to three (14 per cent) at eleven years. Symptomatic instability, pain, and a positive pivot-shift test were the most common reasons for a poor result. Because of the decline in the subjective and objective scores, we no longer recommend the Ellison procedure as the sole operative treatment for a torn anterior cruciate ligament of the knee.


A normal data base of posteroanterior roentgenographic measurements of the wrist. In 120 adults, we measured the dominant wrist on posteroanterior roentgenograms in order to determine the normal dimensions and variations according to age and sex. Men and women were equally represented as were two age-groups (twenty-five to forty years and forty-one to sixty years). The roentgenograms were made, with standard exposure and development techniques, with the wrist and forearm in a neutral position and the x-ray tube aligned vertical to the radial styloid. The roentgenogram that was made with the wrist in the mid-coronal plane was digitized and was used to measure distances between specified landmarks. The mean ulnar variance was -0.9 millimeter (normal range, -4.2 to 2.3 millimeters). The average carpal height ratio was 53 per cent (normal range, 46 to 61 per cent). The mean radial inclination was 24 degrees (range, 19 to 29 degrees). The carpal-radial and carpal-ulnar ratios were smaller in women. The width of the distal radio-ulnar joint was reduced in the older subjects. There was a significant positive relationship (p < 0.0001) between the maximum force reached and the length of the third metacarpal. We believe that information concerning the normal roentgenographic measurements and relationship between the bones of the wrist can be used (1) to follow the progression of carpal instabilities, osteonecrosis, osteoarthrosis, or rheumatoid arthritis; (2) in clinical research; and (3) in the design of wrist implants.


The effects of epinephrine, ibuprofen, and tetrachlorodecaoxide on cutaneous microcirculation in thermally injured hamsters. Fluctuations in the diameter of selected arterioles in the cutaneous microcirculation of Syrian golden hamster dorsal skin flap chambers, which ranged in size from 10 to 70 microns at different branching order sites, were measured before burn, at the same sites after burn and after injection of the drug. Three different drugs epinephrine (administered intravenously), ibuprofen (administered intravenously), and tetrachlorodecaoxide (administered intravenously and topically) were evaluated. Results show that the response to thermal injury in the control group involved extensive vasodilation in the arterioles, prolonged flow irregularities including flow obstructions and stasis, and a decrease in the level of vasoactivity of the microvessels. In two treatment groups, the ibuprofen and tetrachlorodecaoxide groups, significant improvement as indicated by reduced vasodilation and edema and improved microcirculatory blood flow after injury were observed. Further testing of tetrachlorodecaoxide as a topically applied wound dressing is indicated.


Reversible burn injury.

Reversible burn injury. Treatment of the patient with burns has advanced dramatically during the last several decades. Patients with burns that cover a greater percentage of the body surface area are now surviving. Cardiac, pulmonary, and vascular insults are better understood and more adequately treated. Nutritional requirements of the patient with burns can be fulfilled, and physical therapy is providing improved quality of life. In spite of these advances, care of burned tissue has progressed only moderately. The introduction of silver nitrate and other topical antibiotics and early excision significantly improved burn care. Other advances are not in widespread use in spite of research data that detail beneficial alternatives or additions to current therapies. Specifically, reversal of burn injury is possible. The research in this field had progressed considerably to the present. Reversal of dermal vascular occlusion in burned tissue seems to preserve that tissue.


Growth hormone and cortisol secretion in patients with burn injury. A prospective study of growth hormone, insulin-like growth factor (IGF-1), and cortisol secretion was undertaken in six adults with burn injury. Serum concentrations of growth hormone and IGF-1 were low in all patients during the first 2 weeks of hospitalization. The mean growth hormone level was 4.35 +/- 0.83 micrograms/L on day 1 and 1.70 +/- 0.50 micrograms/L on day 13. The mean serum concentration of IGF-1, which reflects overall growth hormone secretion, was 0.43 +/- 0.09 U/ml on day 1 and 0.61 +/- 0.11 U/ml on day 13; these values are distinctly low. After 3 to 4 weeks, IGF-1 concentrations increased to the mid-normal range, whereas growth hormone values did not change. Morning plasma cortisol concentrations were modestly elevated; however, urine free cortisol concentrations, which reflect total cortisol secretion, were elevated 2 to 28 times above normal values at the time of admission (mean, 443.5 +/- 323.7 nmol/L). Urinary free cortisol concentrations remained elevated after 2 weeks (mean, 230.5 +/- 94.5 nmol/L). Patients with burn injury have inappropriately low growth hormone secretion and IGF-1 production in spite of the stress of the injury and more than adequate nutritional therapy.