P-domains as shuffled cysteine-rich modules in integumentary mucin C.1 (FIM-C.1) from Xenopus laevis. Polydispersity and genetic polymorphism. A new frog integumentary mucin (FIM-C.1) has been discovered by molecular cloning. This mucin contains at least six typical P-domains as cysteine-rich modules. Shuffled P-domains have previously been detected in FIM-A.1, and they also form the basis of various P-domain peptides, which presumably have growth-modulating activities. Furthermore, FIM-C.1 contains at least three threonine-rich clusters, which consist of semi-repetitive cassettes. Various polydisperse transcripts have been characterized. They originate from two genes only and differ by deletions/insertions that are congruent with the semi-repetitive cassettes. Thus, polydispersities are probably generated by alternative splicing. Southern analysis revealed genetic polymorphism between different individuals. Furthermore, a specific antiserum was generated against a synthetic peptide deduced from the COOH-terminal end of FIM-C.1 and used for Western analysis.


Two related genes encoding extremely hydrophobic proteins suppress a lethal mutation in the yeast mitochondrial processing enhancing protein. The processing enhancing protein of mitochondria (PEP) is an essential component that has been shown to participate in proteolytic removal of NH2-terminal signal peptides from precursor proteins imported into the mitochondrial matrix. Using a yeast strain bearing a PEP mutation that renders it temperature-sensitive, an approach of genetic suppression was taken in order to identify additional components that could be involved with protein import: high copy plasmids comprising a yeast genomic library were tested for ability to suppress the 37 degrees C growth defect. Two plasmids were isolated, pSMF1 and pSMF2, which suppressed the growth defect nearly as well as the cloned PEP gene itself. Sequence analysis of the rescuing genes predicted extremely hydrophobic proteins with sizes of 63 and 60 kDa, respectively. Remarkably, the predicted SMF1 and SMF2 products are 49% identical to each other overall. To test the requirement for SMF1 and SMF2, the chromosomal genes were disrupted. Individual disruption was without effect, but cells in which both genes were disrupted grew poorly. When mitochondria were prepared from the double disruption strain grown in a nonfermentable carbon source, they were morphologically normal but defective for translocation of radiolabeled precursor proteins. SMF1 protein was provisionally localized to the mitochondrial membranes using epitope tagging. We suggest that SMF1 and SMF2 are mitochondrial membrane proteins that influence PEP-dependent protein import, possibly at the step of protein translocation.


Inactivation of ribonuclease inhibitor by thiol-disulfide exchange. Porcine ribonuclease inhibitor (RI) contains 30 1/2-cystinyl residues, all of which occur in the reduced form. Reaction of the native protein with 5,5'-dithiobis (2-nitrobenzoic acid) resulted in the release of 30 mol of the product 5-mercapto-2-nitrobenzoate, and the loss of the RNase inhibitory activity. A linear relationship between the degree of modification and inactivation was observed. The rate of modification was greatly increased in the presence of 6 M guanidinium HCl. Reaction with substoichiometric amounts of 5,5'-dithiobis(2-nitrobenzoic acid) was found to yield a mixture of fully reduced active molecules, and fully oxidized inactive ones, but no partially oxidized forms were detected. This suggests that an "all-or-none" type of modification and inactivation took place. All 1/2-cystinyl residues in the inactive, monomeric inhibitor had formed disulfide bridges, judged by the absence of either free thiol groups or mixed disulfides with 5-mercapto-2-nitrobenzoate. This fully disulfide-cross-linked molecule had an open conformation compared to the native one, as shown by gel filtration and limited proteolysis. Reaction of phenylarsinoxide with vicinal dithiols yields products that are much more stable than those with monothiols. Titration of RI with this reagent yielded complete inactivation at a reagent/thiol ratio of 0.5. Taken together, these observations suggest that the thiol groups in RI have a diminished reactivity due to three-dimensional constraints. After the initial modification of a small number of thiol groups, a conformational change occurs which causes an increase in reactivity of the remaining thiols. The thiol groups are situated close enough together to permit the formation of 15 disulfide bridges in the inactive molecule.


Mutational analysis of the Escherichia coli phosphate-specific transport system, a member of the traffic ATPase (or ABC) family of membrane transporters. A role for proline residues in transmembrane helices. The Escherichia coli Pst system is a periplasmic phosphate permease. A mutational analysis of the requirement for function of specific charged residues or proline residues in the two hydrophobic subunits (PstC and PstA) has been carried out. No residues, among 19 charged residues altered, were found to be essential for phosphate uptake, although some alterations resulted in partial effects. Evidence was obtained that the 3 residues, R220 in the PstA protein and R237 and E241 in the PstC protein, previously shown to be required for phosphate transport (Cox, G. B., Webb, D., Godovac-Zimmermann, J., and Rosenberg, H. (1988) J. Bacteriol. 170, 2283-2286; Cox, G. B., Webb, D., and Rosenberg, H. (1989) J. Bacteriol. 171, 1531-1534), interact with each other. A feature of the proposed structures of the PstA and PstC proteins was 2 pairs of proline residues in putative transmembrane helices 3 and 4. While individual substitutions of these proline residues by leucine resulted in loss of phosphate transport activity substitution by alanine only had partial effects. However, if the proline to alanine changes were paired then, depending on the particular subunit, markedly different effects were obtained. The double mutation in the PstA protein resulted in a permanently "closed" system, whereas the double mutation in the PstC protein resulted in a permanently "open" transport system.


Characterization of the spectrum of alternative splicing of alpha 1 (XIII) collagen transcripts in HT-1080 cells and calvarial tissue resulted in identification of two previously unidentified alternatively spliced sequences, one previously unidentified exon, and nine new mRNA variants. Amplification of a COL1-encoding region of alpha 1 (XIII) collagen transcripts of HT-1080 cell RNA suggested that exon 3 of the alpha 1 (XIII) collagen gene, which was previously deduced to be of 35 base pairs (bp) may consist of a constitutive 8-bp exon and an alternatively spliced 27-bp exon, termed here exons 3A and 3B, respectively. Furthermore, a previously unidentified alternatively spliced Gly-Xaa-Yaa-encoding exon designated as 4B was found between the sequences encoded by exons 4, redesignated here as 4A and 5. Six of the 16 potential combinations of the four consecutive alternatively spliced exons 3B, 4A, 4B, and 5 were found to exist in mRNAs, and as a result, the length of the COL1 domain may vary between 57 and 104 amino acid residues. Most of the NC2 domain is encoded by the alternatively spliced exons 12 and 13. Where previous analysis of cDNAs indicated that mRNA variants exist that contain either exon 12 or 13 sequences, amplification studies indicated here that there are also variants that lack both exons 12 and 13 but none that contain both exons simultaneously. Thus, the predicted length of this domain is either 12, 31, or 34 residues. Analyses covering both the COL1 and NC2 domains demonstrate that at least 12 mRNA species exist through the alternations of exons 3B-5, 12, and 13.


Patterns of expression of the six alternatively spliced exons affecting the structures of the COL1 and NC2 domains of the alpha 1(XIII) collagen chain in human tissues and cell lines. Reverse transcription-polymerase chain reactions and RNA protection experiments were used to examine alternative splicing of the six exons 3B-5, 12, and 13 affecting the COL1 and NC2 domains of type XIII collagen in seven human tissues and four cell lines. Distinct differences in the proportions of the variant mRNAs were found. With respect to the COL1 domain, all studied samples contained mRNAs corresponding to the shortest COL1 variants of 57 and 66 residues, with the former variant being prominent in most samples. Most of the samples also contained notable amounts of mRNAs that corresponded to the longest COL1 variants, mainly those of 104 and 95 residues. Particularly the extent of inclusion of exon 12 and 13 sequences, encoding most of the NC2 domain, varied according to the type of tissue or cell analyzed. Bone, cartilage, and colon adenocarcinoma samples contained little or none of the mRNAs corresponding to the long NC2 variants, whereas in fibroblast, lung, muscle, and osteosarcoma cells, those mRNAs were the major variants. The relative proportions of the various combinations of exons 3B-5, 12, and 13 were evaluated in four of the RNA samples. Interestingly, each of these samples appeared to contain only one to three major combinations of the six exons, representing about 40% to nearly 100% of all variants.


Isolation and characterization of the yeast gene encoding the MDH3 isozyme of malate dehydrogenase. The MDH3 isozyme of Saccharomyces cerevisiae was purified from a haploid strain containing disruptions in genomic loci encoding the mitochondrial MDH1 and nonmitochondrial MDH2 isozymes. Partial amino acid sequence analysis of the purified enzyme was conducted and used to plan polymerase chain reaction techniques to clone the MDH3 gene. The isolated gene was found to encode a 343-residue polypeptide with a molecular weight of 37,200. The deduced amino acid sequence was closely related to those of MDH1 (50% residue identity) and of MDH2 (43% residue identity). The MDH3 sequence was found to contain a carboxyl-terminal SKL tripeptide, characteristic of many peroxisomal enzymes, and immunochemical analysis was used to confirm organellar localization of the MDH3 isozyme. Levels of MDH3 were determined to be elevated in cells grown with acetate as a carbon source, and under these conditions, MDH3 contributed approximately 10% of the total cellular malate dehydrogenase activity. Disruption of the chromosomal MDH3 locus produced a reduction in cellular growth rates on acetate, consistent with the presumed function of this isozyme in the glyoxylate pathway of yeast. Combined disruption of MDH1, MDH2, and MDH3 loci in a haploid strain resulted in the absence of detectable cellular malate dehydrogenase activity.


A comparison of the rates of reaction and function of UVRB in UVRABC- and UVRAB-mediated anthramycin-N2-guanine-DNA repair. The repair of anthramycin-DNA adducts by the UVR proteins in Escherichia coli follows two pathways: the adducts may be incised by the combined actions of UVRA, UVRB, and UVRC, or alternatively, the anthramycin may be removed by UVRA and UVRB in the absence of UVRC and with no DNA strand incision. To assess the competition between these two competing pathways, the rate of UVRABC-mediated excision repair of anthramycin-N2-guanine DNA adducts and the rate of UVRAB-mediated removal of the adduct were measured with single end-labeled DNAs under identical reaction conditions. UVR protein concentrations of 15 nM UVRA, 100 nM UVRB, and 10 nM UVRC protein were chosen to mimic in vivo concentrations. With these UVR protein concentrations and anthramycin-DNA concentrations of 1-2 nM the incision reaction and the release reactions are described by first-order kinetics. The rate of the UVRABC reaction, measured as the increase in incised fragments, was six to seven times faster than the rate of the UVRAB reaction, measured as the decrease in incised fragments. The UVRABC incision rate on anthramycin-modified linear DNA was four to five times the incision rate measured on the same DNA irradiated with ultraviolet light. We also investigated the role of the ATPase function of UVRB in UVRAB-mediated anthramycin removal. We found that a UVRB analogue with alanine at arginine 51, which retains near wild type ATPase activity, supported removal of anthramycin in the presence of UVRA, whereas a UVRB analogue with alanine at lysine 45, which abolishes the ATPase activity, did not. UVRB*, a specific proteolytic cleavage product of UVRB which retains the ATPase activity, did support removal of anthramycin in the presence of UVRA.


Cell surface expression of the C3b/C4b receptor (CR1) protects Chinese hamster ovary cells from lysis by human complement. The C3b/C4b receptor, also known as complement receptor type 1 (CR1, CD35), is a single chain glycoprotein consisting of 30 repeating homologous protein domains known as short consensus repeats (SCR) followed by transmembrane and cytoplasmic domains. A series of recombinant proteins derived from CR1 has been prepared and assessed for the capacity to inhibit complement lysis of the host Chinese hamster ovary (CHO) cells. The full-length recombinant CR1 inhibited human complement-mediated CHO cell lysis, and the efficiency of inhibition was directly proportional to the number of receptors/cell. The SCR 15-18 of CR1, but not SCR 15-16, inhibited complement lysis of the host CHO cell, bound monomeric C3b (Kd,app = 6.5 x 10(-7) M), and dimeric C3b (Kd = 1.8 x 10(-8) M), and served as a cofactor in the proteolysis of C3b by factor I, confirming and extending the observations of Fearon and colleagues (Kalli, K. R., Hsu, P., Bartow, T. J., Ahearn, J. M., Matsumoto, A. K., Klickstein, L. B., and Fearon, D. T. (1991) J. Exp. Med. 174, 1451-1460). The SCR 1-4 of CR1, but not SCR 1-2, also inhibited complement lysis of the host CHO cell, indicating that more than two SCR are necessary and that four SCR are sufficient for optimal C4b binding to CR1. Thus, the structural requirements for C4b binding are analogous to those for C3b binding, namely, four SCR of CR1 form the binding sites for each of these proteins. CR1 has long been recognized to regulate extrinsic complement activation, that is, to bind to and promote the degradation of fluid phase C3b and of C3b attached to immune complex. These results demonstrate that CR1 is also an intrinsic regulator of complement activation in that, under appropriate conditions, CR1 inhibits complement-mediated lysis of the cell on which it is expressed.


A role for the acidic trimer repeat region of transcription factor sigma 54 in setting the rate and temperature dependence of promoter melting in vivo. sigma 54 is an atypical sigma factor involved in enhancer-dependent transcription in Escherichia coli. In vivo assays were developed for following the kinetics and thermodynamics of sigma 54-dependent melting of the glnAP2 promoter start site. These assays were applied to a series of three sigma 54 polymerases containing zero, one, and two copies of the highly acidic trimer repeat region. The results showed that at least one copy of this acid region is required for a complete melting transition at physiological temperature, but is not required for a low temperature melting transition. The rate of melting the promoter in vivo increased with number of copies of this region. Taken together with other observations, the experiments point to a role for the acid region in triggering conformational changes that lead to specific promoter melting. The acid region is required for these changes to occur fully at physiological temperature and influences the rate at which they occur.


A third form of the G protein beta subunit. 1. Immunochemical identification and localization to cone photoreceptors. Vertebrate retinal cones play a major role in both photopic vision and color perception. Although the molecular mechanism of visual excitation in the cone is not as well understood as in the rod, it is generally thought to involve a cone-specific G protein (cone transducin) that couples the cone visual pigment to a cGMP phosphodiesterase. Like all other G proteins, cone transducin is most likely a heterotrimer consisting of G alpha, G beta, and G gamma subunits. A G alpha subunit of cone transducin has been localized to the outer segment of bovine cones, but its associated G beta and G gamma subunits are unknown. To identify the G beta subunit involved in the phototransduction process of cones, we have developed a panel of antipeptide antisera against the most diverse region of the amino acid sequences encoded by G beta 1, G beta 2, and G beta 3 cDNAs and used them to determine the distribution of the G beta isoforms in different retinal preparations. We found that the G beta 3 subunit is present in bovine retinal transducin and phosducin-T beta gamma complex preparations which were previously thought to contain only G beta 1. Analysis of its subcellular distribution indicated that G beta 3 is predominantly cytoplasmic. Immunocytochemical staining of bovine retinal sections with the anti-G beta 3 antiserum further revealed a specific localization of G beta 3 in cones but not in rods. In contrast, anti-G beta 1 antiserum stained only the rods. These results suggest that G beta 3 is the G beta subunit of cone transducin and confirms the proposition that rods and cones utilize distinct signaling proteins for phototransduction.


Modulation of a Ca2+ signaling pathway by GM1 ganglioside in PC12 cells. The effects of exogenous GM1 ganglioside on depolarization and ligand-induced Ca2+ signaling were investigated in PC12 cells. Cellular responses to K+ depolarization and bradykinin application in control and GM1-treated cells were examined with respect to: 1) changes in the intracellular Ca2+ concentration ([Ca2+]i) measured using fura-2 fluorescence in single cells, and 2) changes in Ca(2+)-dependent protein kinase activity as assayed by two-dimensional phosphopeptide analysis of the site-specific phosphorylation of tyrosine hydroxylase. Pretreatment of cells with GM1 (10 or 100 microM) enhanced K+ depolarization-stimulated increases in [Ca2+]i and in 32PO4 incorporation into tyrosine hydroxylase phosphopeptide T2, a Ca2+/calmodulin-dependent protein kinase II substrate. In contrast, GM1 treatment had no effect on the transient increases in [Ca2+]i evoked by bradykinin or on bradykinin-induced increases in the site-specific phosphorylation of tyrosine hydroxylase. The depolarization-induced and GM1-enhanced increases in [Ca2+]i and T2 phosphorylation were prevented by removal of external Ca2+ or pretreatment with 1 microM nitrendipine, suggesting that these increases result from Ca2+ entry through dihydropyridine-sensitive Ca2+ channels. The ability of exogenous gangliosides to potentiate increases in [Ca2+]i may underlie their diverse neuritogenic and neurotrophic actions in the nervous system.


Structure and chromosomal localization of human arachidonate 12-lipoxygenase gene. Arachidonate 12-lipoxygenase introduces a molecular oxygen into the C-12 position of arachidonic acid to produce 12(S)-hydroperoxy-5,8,10,14-eicosatetraenoic acid. With the aid of cDNA probes for the enzyme, we isolated overlapping lambda clones encompassing the human 12-lipoxygenase gene and flanking regions from a human genomic library. The gene consists of 14 exons with 13 introns and spans approximately 15 kilobases of DNA. All the exon-intron junctions conform to the GT/AG rule. Neither a typical TATA box nor a CAAT box was found in approximately 1-kb sequence of 5'-upstream region of the translation initiation site. However, this region contains several regulatory elements including four GC boxes, two CACCC boxes, three AP-2 binding sequences, and a glucocorticoid-responsive element. The major transcription initiation site was determined by primer-extension analysis as an adenosine residue at 306 bases upstream from the translation initiation codon. The chromosomal localization of the human 12-lipoxygenase gene was examined by fluorescence in situ hybridization, and the gene was assigned to the sub-band p13.1 of chromosome 17.


Stereoselective interaction with chiral phosphorothioates at the central DNA kink of the EcoRI endonuclease-GAATTC complex. We have probed the contacts between EcoRI endonuclease and the central phosphate of its recognition site GAApTTC, using synthetic oligonucleotides containing single stereospecific Rp- or Sp-phosphorothioates (Ps). These substitutions produce subtle stereospecific effects on EcoRI endonuclease binding and cleavage. An Sp-Ps substitution in one strand of the DNA duplex improves binding free energy by -1.5 kcal/mol, whereas the Rp-Ps substitution has an unfavorable effect (+0.3 kcal/mol) on binding free energy. These effects derive principally from changes in the first order rate constants for dissociation of the enzyme-DNA complexes. The first order rate constants for strand scission are also affected, in that a strand containing Sp-Ps substitution is cleaved 2 to 3 times more rapidly than a strand containing a normal prochiral phosphate, whereas a strand containing Rp-Ps substitution is cleaved about 3 times slower than normal. As a result, single-strand substitutions produce pronounced asymmetry in the rates of cleavage of the two DNA strands, and this effect is exaggerated in an Rp,Sp-heteroduplex. Ethylation-interference footprinting indicates that none of the Ps substitutions cause any major change in contacts between endonuclease and DNA phosphates. When an Sp-Ps localizes P = O in the DNA major groove, a hydrogen-bonding interaction with the backbone amide-NH of Gly116 of the endonuclease is improved relative to that with a prochiral phosphate having intermediate P-O bond order and delocalized charge.


An atomic model for protein-protein phosphoryl group transfer. The high resolution crystal structures of two interacting proteins from the phosphoenolpyruvate:sugar phosphotransferase system, the histidine-containing phosphocarrier protein (HPr) and the IIA domain of glucose permease (IIA(Glc)) from Bacillus subtilis, provide the basis for modeling the transient binary complex formed during the phosphoryl group transfer. The complementarity of the interacting surfaces implies that no major conformational transition is required. The negatively charged phosphoryl group is buried in the interface, suggesting a key role for electrostatic interactions. It is proposed that the phosphoryl transfer is triggered by a switch between two salt bridges involving Arg-17 of the HPr. The first, prior to phosphoryl group transfer, is intramolecular, with the phosphorylated His-15. The second, during the transfer, is intermolecular, with 2 aspartate residues associated with the active site of IIA(Glc). Such alternating ion pairs may be mechanistically important in other protein-protein phosphotransfer reactions.


Use of structure-directed DNA ligands to probe the binding of recA protein to narrow and wide grooves of DNA and on its ability to promote homologous pairing. We have used circular dichroism and structure-directed drugs to identify the role of structural features, wide and narrow grooves in particular, required for the cooperative polymerization, recognition of homologous sequences, and the formation of joint molecules promoted by recA protein. The path of cooperative polymerization of recA protein was deduced by its ability to cause quantitative displacement of distamycin from the narrow groove of duplex DNA. By contrast, methyl green bound to the wide groove was retained by the nucleoprotein filaments comprised of recA protein-DNA. Further, the mode of binding of these ligands and recA protein to DNA was confirmed by DNaseI digestion. More importantly, the formation of joint molecules was prevented by distamycin in the narrow groove while methyl green in the wide groove had no adverse effect. Intriguingly, distamycin interfered with the production of coaggregates between nucleoprotein filaments of recA protein-M13 ssDNA and naked linear M13 duplex DNA, but not with linear phi X174 duplex DNA. Thus, these data, in conjunction with molecular modeling, suggest that the narrow grooves of duplex DNA provide the fundamental framework required for the cooperative polymerization of recA protein and alignment of homologous sequences. These findings and their significance are discussed in relation to models of homologous pairing between two intertwined DNA molecules.


The crystal structure of the aldose reductase.NADPH binary complex. Aldose reductase is an NADPH-dependent oxidoreductase that catalyzes the reduction of a broad range of aldehydes, including glucose. Since aldose reductase has been strongly implicated in the development of the chronic complications of diabetes mellitus, much effort has been devoted to understanding the structure and mechanism of this enzyme, and many aldose reductase inhibitors have been developed as potential drugs for the treatment of these complications. We describe here the 2.75 A crystal structure of recombinant human aldose reductase (Cys-298 to Ser mutant) complexed with NADPH. This mutant displays unusual kinetic behavior characterized by high Km/high Vmax substrate kinetics and reduced sensitivity to certain aldose reductase inhibitors. The crystal structure revealed that the enzyme is a beta/alpha-barrel with the coenzyme-binding domain located at the carboxyl-terminal end of the parallel strands of the barrel. The enzyme undergoes a large conformational change upon binding NADPH which involves the reorientation of loop 7 to a position which appears to lock the coenzyme into place. NADPH is bound to aldose reductase in an unusual manner, more similar to FAD- rather than NAD(P)-dependent oxidoreductases. No disulfide bridges were observed in the crystal structure.


AraC protein contacts asymmetric sites in the Escherichia coli araFGH promoter. AraC protein regulates the transcription of arabinose transport and catabolic operons in Escherichia coli through interaction with specific DNA sequences in the promoter regions of the operons. The interaction of AraC protein with two binding sites in the araFGH promoter was determined and compared to previously studied AraC binding sites in the araBAD promoter. Methylation and ethylation interference assays show that AraC protein binds along one side of the DNA to four adjacent major groove regions at each of the araFG1 and araFG2 sites. Mutations within any of the four regions of araFG1 greatly reduce protein binding in vitro. The promoter function in vivo is also greatly reduced, indicating that all four regions of the binding site are required. The chemical interference and genetic data, combined with the consensus sequence for AraC protein binding to ara promoters, support a binding motif in which two directly repeated units each span two adjacent turns of the DNA helix. The function of the two AraC binding sites was also examined. The proximal araFG1 site is required for promoter activation, whereas the distal araFG2 site has only a slight effect on the promoter activity.


Calcium and collagen binding properties of osteopontin, bone sialoprotein, and bone acidic glycoprotein-75 from bone. Calcium binding properties of bone acidic glycoprotein-75, osteopontin, and bone sialoprotein were determined in 10 mM imidazole buffer (pH 6.8), containing either 60 mM KCl or 150 mM NaCl. Proteins assayed were first bound to nitrocellulose to mimic substrate-bound forms in vivo; retention of phosphoproteins was determined through use of radioiodinated tracers. Binding studies were carried out both as a function of calcium concentration and the amount of phosphoprotein. In the presence of 60 mM KCl, bone acidic glycoprotein-75 exhibited the largest calcium binding capacity (139 atoms/molecule at saturation), with bone sialoprotein intermediary (83 atoms/molecule) and osteopontin lowest (50 atoms/molecule). Sites detected for each phosphoprotein exhibited overall binding constants in the 0.5-1.0 mM extracellular range. In 150 mM NaCl and 1-2 mM total calcium, phosphoproteins bound between 72 and 19 mol of calcium/mol with the same relative order. Binding was proportional to amount of phosphoprotein in either salt condition. The presence of 5 mM calcium had a different effect on concentration-dependent binding to type I collagen for each phosphoprotein. Bone acidic glycoprotein-75 alone was found to undergo an unusual calcium-enhanced polymerization reaction, confirmed by light scattering measurements, wherein collagen binding was greatest with polymeric forms. These findings demonstrate that acidic phosphoproteins from bone bind calcium atoms with a range of capacities. Calcium appears to induce conformational changes in bone acidic glycoprotein-75 which influences its self-association and binding to different substrata.


Induction of heparin-binding epidermal growth factor-like growth factor mRNA by phorbol ester and angiotensin II in rat aortic smooth muscle cells. To determine whether the gene encoding the recently identified heparin-binding epidermal growth factor-like growth factor (HB-EGF), a potent smooth muscle cell (SMC) mitogen of macrophage origin, is transcribed and regulated in vascular SMC, we isolated cDNA clones encoding rat HB-EGF from a macrophage library. Using the rat HB-EGF cDNA as a probe for RNA blot analysis, we detected low levels of HB-EGF mRNA in rat aortic SMC in culture. However, 20 nM 12-O-tetradecanoylphorbol-13-acetate (TPA) and 10(-6) M angiotensin II (AII) induced a marked increase in HB-EGF mRNA levels in rat aortic SMC (11- and 4.6-fold, respectively) that was both dose- and time-dependent. In response to TPA and AII, HB-EGF mRNA levels increased rapidly, peaked at 2 h, and returned to base line at 7 h. This effect of AII on HB-EGF induction was specific, as evidenced by the fact that it could be completely blocked by the AII antagonist saralasin. This is the first demonstration that HB-EGF is transcribed and regulated in SMC. The inducible transcription of this potent SMC mitogen gene in vascular SMC suggests that HB-EGF may have an important autocrine role in the proliferation of SMC in vascular diseases such as atherosclerosis and hypertension.


Dissociation of the contractile and hypertrophic effects of vasoconstrictor prostanoids in vascular smooth muscle. To more clearly define the physiologic roles of thromboxane (TX)A2 and primary prostaglandins (PG) in vascular tissue we examined vascular contractility, cell signaling, and growth responses. The growth-promoting effects of (15S)-hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5Z,13E-dienoic acid (U46619; TXA2 agonist), PGF2 alpha, and PGE2 consisted of protein synthesis and proto-oncogene expression, but not DNA synthesis or cell proliferation. U46619 contracted rat aortas and increased cultured rat aortic vascular smooth muscle cell intracellular free calcium concentration [Ca2+]i, [3H]inositol monophosphate (IP) accumulation, myosin light chain phosphorylation, and protein synthesis ([3H]leucine incorporation) with EC50 values ranging from 10 to 50 nM. Each of these responses was inhibitable with the TXA2 receptor antagonist [1S]1 alpha,2 beta(5Z),3 beta,4 alpha-7-(3-[2- [(phenylamino)carbonyl]hydrazino]methyl)-7-oxabicyclo[2.2.1]hept-2- yl-5-heptenoic acid (SQ29548). In contrast, PGF2 alpha increased [Ca2+]i, [3H]IP, and protein synthesis with EC50 values of 30-230 nM but contracted rat aortas with an EC50 of 4800 nM. PGE2 increased [Ca2+]i, [3H]IP accumulation, protein synthesis, and contracted rat aortas with EC50 values of 2.5-3.5 microM. TXA2 receptor blockade prevented PGF2 alpha- and PGE2-induced aortic contraction and cell myosin light chain phosphorylation, but not cell signaling or protein synthesis. Binding studies to vascular smooth muscle TXA2 receptors using 1S-[1 alpha,2 beta(5Z),3 alpha(1E,3S),4 alpha]-7-(3-[3-hydroxy-4-(p- [125I]iodophenoxy)-1-butenyl]7-oxabicyclo[2.2.1]hept-2-yl)-5-hepte noic acid ([125I]BOP) showed U46619, SQ29548, PGF2 alpha, and PGE2 competition for TXA2 receptor binding at concentrations similar to their EC50 values for aortic contraction, while binding competition with [3H]PGF2 alpha and [3H]PGE2 demonstrated the specificity of [125I]BOP and SQ29548 for TXA2 receptors. The results suggest that 1) PGF2 alpha- and E2-stimulated vessel contraction is due to cross-agonism at vascular TXA2 receptors; 2) PGF2 alpha stimulates TXA2 receptor-independent vascular smooth muscle protein synthesis at nanomolar concentrations, consistent with an interaction at its primary receptor; and 3) TXA2 is a potent stimulus for vascular smooth muscle contraction and protein synthesis. We suggest that the main physiologic effect of PGF2 alpha may be as a stimulus for vascular smooth muscle cell hypertrophy, not as a contractile agonist.


Sphingolipid transport in mitotic HeLa cells. Mitotic and interphase HeLa cells were labeled with [3H]serine. Ceramide and its derivatives, lactosylceramide and sphingomyelin, were biosynthetically labeled under both conditions. Only in the absence of nocodazole, as the cells entered telophase, was an additional glycosphingolipid synthesized which was identified as GA2 (GalNAc(beta 1,4)Gal(beta 1,4)Glc(beta 1,1)Cer). Ceramide, the basic sphingolipid precursor, is synthesized in the endoplasmic reticulum, whereas its immediate derivatives are synthesized in early Golgi compartments. Transport of newly synthesized proteins from the endoplasmic reticulum to the Golgi is inhibited in mitotic cells while ceramide acquires early Golgi modifications under the same conditions, suggesting that ceramide can be delivered to the Golgi by a different route. Since GA2 is synthesized in late Golgi, its absence in mitotic cells strongly argues for an in vivo inhibition of intra-Golgi transport, an observation with important implications for the mechanism of Golgi division.


A comparative study of ultrastructures of the interfaces between four kinds of surface-active ceramic and bone. The interfaces between four kinds of surface-active ceramic and bone were studied by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) using undecalcified specimens. The materials were Bioglass-type glass (Bioglass), Ceravital-type glass-ceramic (KGS), apatite- and wollastonite-containing glass-ceramic (A-W.GC) and hydroxyapatite (HA). Particles of these materials, ranging between about 100 and 300 microns in diameter, were implanted into rat tibiae, and specimens were prepared for observation at 8 weeks after implantation. All materials were observed to bond to bone through a collagen-free layer consisting of fine apatite crystals distinct from those in bone. The crystals of this apatite layer and those of bone were intermingled at their interface, suggesting chemical bonding. In Bioglass, which had only a glassy phase, several tens of microns of the material surface had changed to such an apatite layer. In KGS and A-W.GC, which had macrocrystals in the glassy phase, an intervening apatite layer about 0.5 micron thick was observed between the materials and bone. Furthermore, fine apatite crystals were also observed among the macrocrystals near the surface of the materials. In HA, which had no glassy phase, an intervening apatite layer was much less distinct and sometimes absent. These differences were considered to be attributable to the differences in chemical composition, crystallization, and solubility of the materials.


Influence of fibril length upon ePTFE graft healing and host modification of the implant. Influence of fibril length (porosity) upon synthetic vascular graft healing has not been investigated in detail. The purpose of this study was to determine the dependence of neoendothelial healing, cellular response, and biocompatibility on the fibril length of expanded polytetrafluoroethylene (ePTFE) grafts with an internal diameter of 1.5 mm. ePTFE grafts of different fibril length, 20, 40, 60, and 90 microns, were implanted into the abdominal aorta of rats (n = 5 for each group). After 5 weeks, the implants were harvested and examined for neointimal and pseudointimal coverage by light microscopy and SEM. The hydroxyproline content of the implants was measured, and the distribution of collagen types was examined. The neointimal and pseudointimal coverage was related to the fibril length, and the neoendothelial healing was better on 60-microns and 90-microns grafts than on 20-microns and 40-microns grafts. The amount of hydroxyproline was also related to the fibril length, however, no significant difference could be observed between 60-microns and 90-microns grafts. Collagen types I and III were almost identically located in the middle portion of the implants. Our results demonstrate that the fibril length of ePTFE grafts affected neoendothelial healing and its affinity to collagen.


Spatial and temporal changes in compliance following implantation of bioresorbable vascular grafts. Compliance matching between the host vessel and vascular grafts used for small-diameter arterial replacements is thought to be important for long-term patency. However, currently available grafts elicit fibroplastic reactions, resulting in decreasing compliance with time after implantation. Bioresorbable prostheses elicit ingrowth of myofibroblasts containing abundant contractile elements. This led us to investigate whether compliance of implanted bioresorbable prostheses decreased as a function of time and if the kinetics of change correlated with the progression of tissue ingrowth. Woven polyglactin 910 prostheses (10 mm x 4 mm i.d.) were implanted into adult NZW rabbit infrarenal aortas and replicates were harvested serially through 8 months. Control grafts were implanted, and immediately resected. Dynamic compliance was measured at 1-mm axial increments along each explant using a pulse duplicator apparatus which exposed the harvested samples to realistic pulsatile hemodynamics. Compliance was calculated for proximal, mid, and distal segments of each graft and averaged at each time point by grouping into control (zero time, n = 3), early (1-4 weeks, n = 13), and late (6-36 weeks, n = 9) explant periods. At late explant periods both proximal and distal compliance were significantly greater than mid graft compliance (p < .02 and p < .03, respectively). There was a significant increase in proximal compliance between early and late explant times (p < .01). Measured increases in mid and distal segment compliance over time did not reach statistical significance. Myofibroblast laden tissue ingrowth into the inner capsule followed macrophage phagocytosis and was nearly complete prior to the time that an increase in compliance was demonstrated. Thus since the major histologic episodes precede the change in compliance, these are not likely initiated by this biomechanical change. We hypothesize the graft resorption coupled with the ingrowth of more compliant tissue likely leads to the increased compliance of the graft material.


Toxic effect of ethylene-oxide-sterilized freeze-dried bone allograft on human gingival fibroblasts. Freeze-dried bone allograft (FDBA) which either had or had not been sterilized by exposure to ethylene oxide (EtO) prior to lyophilization was obtained from two commercial sources. EtO-sterilized FDBA was reexposed to EtO as a positive control. Gas chromatograph assays revealed that three out of four commercially obtained EtO sterilized FDBA had no detectable EtO, with one sample having 0.21 parts per million (PPM). Surprisingly, 0.24 PPM was detected in one sample which had not been sterilized with EtO gas. This was presumed due to contamination from a gas-sterilized rubber stopper. In the cell toxicity study, FDBA and human gingival fibroblasts (HGF) were added simultaneously, incubated for 72 h, and fixed and stained. Samples of FDBA sterilized with EtO which were free of EtO did not alter HGF growth. However, the positive control FDBA which contained 0.72 PPM EtO had a deleterious effect on HGF. FDBA with EtO residuals caused morphologic change in HGF.


Porous polyurethane vascular prostheses with variable compliances. A new technique for the preparation of porous vascular prostheses was investigated. Polyurethane solution (5 to 15 wt%) was injected into a mold. After freezing at low temperature (0 degrees C-196 degrees C), solvents were dissolved out with water at 0 degrees C to form porous tubes. The average pore size (several microns to 70 microns), pore occupation (10% to 51%), and compliance (3% to 35%) were easily changed by changing polyurethane concentration, freezing temperature, and freezing methods. Compliances could be decreased gradually by heat treatment. This technique can give a proper pore size (30-60 microns) for tissue ingrowth, and a suitable compliance for matching with arteries and veins. This method might give a desired compliant graft for artificial implantation with the presently valid medical polymers.


Storage and elimination of titanium, aluminum, and vanadium salts, in vivo. Hamsters were injected with titanium, aluminum, and vanadium salts either intraperitoneally or intramuscularly to study the transport, storage, and elimination of these metals. Blood samples were taken at 4 h or 24 h, and urine samples were taken at 24, 48, and 72 h. The hamsters were then injected weekly for 5 weeks after the initial injection. Blood and portions of the kidneys, liver, lung, and spleen were taken at sacrifice. All samples were analyzed for titanium, aluminum, and vanadium concentrations using graphite furnace atomic absorption spectroscopy (GFAAS). Titanium was found not to be excreted in the urine, was found in low levels in the blood, and was elevated over control in the kidney, liver, and spleen. Aluminum detection via GFAAS showed wide standard deviations and high levels in controls; however, aluminum was found to be excreted in the urine, and to be transported by the blood in the experimental animals. A small amount accumulated in the liver and spleen. Vanadium was excreted in high levels in the urine. A small amount was found in the blood, and the level in the organs was below the reliable detection limits. The rapid excretion of vanadium might be related to its solubility in physiological conditions, while the limited excretion of titanium may be related to its being insoluble in the physiologic environment.


Tissue compatibility of collagen-silicone composites in a rat subcutaneous model. Collagen-silicone composites were fabricated and tested for biocompatibility by subcutaneous implantation in rats. The silicone component consisted of addition cure or condensation cure sheets. The collagen component was either (a) a sponge layer 2 mm thick, (b) a thin film 12-20 microns thick, or (c) residual collagen bonded to or incorporated in the silicone rubber. Collagen sponges were mechanically bonded to silicone sheets, and collagen thin films and residual collagen were physically and chemically attached to epoxy-derivatized silicone sheets. Analysis of implanted samples showed that reduced capsule formation occurred around collagen sponge-silicone, compared to control silicone sheets. Only where the underlying silicone sheet, or interpenetrating silicone, was exposed to the tissue, did limited capsule formation occur. In contrast, thin capsule developed completely around silicone coated with a thin collagen film and around silicone bonded to residual collagen. Sponge-silicone composites and control silicone sheets were free of acute and chronic inflammation, except for occasional foreign body giant cells in sponge adjacent to silicone. Silicone coated with micron-thick collagen films exhibited some inflammation, but residual collagen-silicone did not. This study suggests that, to prevent capsule formation, a collagen coat must be of minimum thickness and surface coverage sufficient to prevent any contact between silicone and tissue.


The significance of calf thrombi after total knee arthroplasty. We reviewed the records of 1257 patients having 1625 total knee arthroplasties; all had pre-operative and postoperative perfusion lung scans and postoperative venograms which were classified as showing no thrombi, calf thrombi or proximal thrombi. Patients with calf thrombi were found to have a significantly greater risk for both symptomatic and asymptomatic pulmonary embolism compared with patients with no venographic thrombi. There were positive lung scans in 6.9% of patients with calf thrombi compared with 2.0% of patients with negative venograms (p < 0.001). Symptomatic pulmonary embolism occurred in 1.6% of patients with calf thrombi compared with 0.2% of patients with negative venograms (p = 0.034). The risk of pulmonary embolism was not significantly different between patients with treated proximal thrombi, and those with calf thrombi. Patients who develop deep-vein thrombosis despite prophylaxis are at increased risk for pulmonary embolism; these patients should receive treatment, or undergo follow-up studies to detect proximal propagation.


Long-term results of total condylar knee arthroplasty in rheumatoid arthritis. We have reviewed at an average period of ten years the results of 71 consecutive primary arthroplasties with the Insall-Burstein total condylar knee prosthesis in patients with rheumatoid arthritis. Their mean age at surgery was 52 years (24 to 72). At follow-up the overall results (Hospital for Special Surgery knee rating score) were excellent or good in 77%, fair in 11% and poor in 11%. There was residual pain in only 5% of patients with prostheses in situ; 58% could walk more than 500 m, and the median range of motion was 108 degrees. Eight knees had been revised. Five underwent arthrodesis because of deep infection and three needed revision arthroplasty for mechanical loosening. The crude survival rate of the arthroplasties was 89%. The presence of radiolucency around the tibial component correlated significantly with the severity of residual pain.


Soft-tissue balance and alignment in medial unicompartmental knee arthroplasty. We reviewed two similar groups of patients with medial osteoarthritis of the knee treated by unicompartmental arthroplasty. The group receiving an Oxford meniscal-bearing implant, with no medial release, showed significantly better mechanical alignment than that receiving a fixed-bearing implant. Under-correction, with its ominous mechanical implications, was much more common with the fixed-bearing design. Over-correction was rare and was seen in both designs about equally. Degenerative stenosis of the intercondylar notch was common and appeared to put the anterior cruciate ligament at risk of rupture, especially after correction of the varus deformity. We consider that postoperative leg alignment and soft-tissue balance after unicompartmental knee replacement are determined more by the implant design and the surgical technique than by any variation in soft-tissue contracture. Release of the medial collateral ligament is not necessary for realignment, but a generous notchplasty is often needed to allow normal anterior cruciate ligament function.


Isolated rupture of the popliteus with posterior tibial nerve palsy. We report the case of a 59-year-old man with severe knee pain and inability to flex his toes or invert his plantar flexed foot after an external rotation injury to his knee. MRI showed rupture of the popliteus with a haematoma compressing the neurovascular bundle in the proximal calf, and electromyography demonstrated signs of an axonotmesis of the posterior tibial nerve. There was progressive nerve recovery over 24 weeks. Isolated rupture of the popliteus should be considered in any patient with an acute haemarthrosis, lateral tenderness and a stable knee, especially after an external rotation injury.


A collarless cobalt-chrome femoral component in uncemented total hip arthroplasty. Five- to eight-year follow-up. We implanted 57 uncemented cobalt-chrome porous-coated collarless femoral components into 51 patients (mean age 49 years). At review, five to eight years postoperatively, good or excellent results were recorded in 70% by the Mayo Clinic hip evaluation and in 84% by the Harris hip score. Revision for aseptic loosening of the femoral stem was necessary in only one hip. Thigh pain diminished with time and was present in only two hips at the time of review. Endosteal bone formation was seen at the junction of the smooth and the porous segments of the stem in 94% of hips and in 60% it continued after three years. In 90% of hips, proximal femoral atrophy did not progress after three years. Discontinuous radiolucent lines were seen around 30% of stems, most commonly in zones I, IV and VII. They were not progressive in 94% and their presence did not correlate with the clinical outcome.


Cemented versus uncemented femoral components in the ring hip prosthesis. A prospective, randomised and independently assessed trial of the Ring UPM total hip replacement showed that the quality of the early result was better if the femoral prosthesis was cemented than if it was not. More patients with cemented prostheses were painfree at four months (58% cemented:42% uncemented) and at one year (63% cemented:50% uncemented), but at two years pain relief was equal in both groups. At two years significantly more patients with cemented prostheses could walk without support (96% cemented:62% uncemented, p = 0.01 to 0.05). There is a need for more similar trials to compare the results of contemporary designs of cemented and uncemented total hip prostheses.


Lymphocyte response to polymethylmethacrylate in loose total hip prostheses. We investigated the lymphocyte-mediated immune response to polymethylmethacrylate bone cement in 26 patients who had revision surgery for aseptic loosening of cemented total hip arthroplasties, at a mean time of seven years after the first replacement. We studied eight patients with cemented total hip arthroplasties which were not loose as controls. Patch tests to polymethylmethacrylate bone cement were positive in 13 patients with loosening, and these patients had higher lymphoblast transformation values against polymethylmethacrylate bone cement patients with a negative skin reaction (p < 0.01) or those in the control group (p < 0.001). Specific monoclonal antibodies were used to assess the percentage of certain cells of the immune system according to their cluster of differentiation (CD). There was a higher number of total T and B lymphocytes (CD2 and CD22) and interleukin-2 receptor-positive lymphocytes (activated cells, CD25) in patients with loose prostheses. More CD25 lymphocytes were found in patients with positive patch tests. The activation of the lymphocyte-mediated immune response was not related to the presence or absence of aggressive granulomatous lesions at the cement-bone interface.


Systemic distribution of wear debris after hip replacement. A cause for concern? The production of particulate wear debris is a recognised complication of joint arthroplasty, but interest has concentrated on local tissue reactions and a possible association with implant loosening. The fate of wear products in the body remains unknown, although some of the metals used in the construction of orthopaedic implants are known to have toxic and oncogenic properties. We report histological and electron-microscopic evidence from two cases which shows that metallic debris can be identified in the lymphoreticular tissues of the body distant from the hip some years after joint replacement. The increase in the use of total arthroplasty in younger patients, the development of new alloys and the use of porous coatings must raise concern for the long-term effects of the accumulation of wear debris in the body.


Classification of fractures of the tibial condyles. We analysed 131 fractures of the tibial condyles in 130 patients, using a modification of the classification of Schatzker, McBroom and Bruce (1979). The patients were reviewed at an average of 7.6 years after the injury. Fifty-five (42%) fractures had been treated conservatively and 76 (58%) operatively. Medial unicondylar and medially tilted bicondylar fractures tended to redisplace into varus position and lateral unicondylar and laterally tilted bicondylar fractures into valgus. There were significant differences when the results were evaluated according to the methods of Hohl and Luck (1956) and Rasmussen (1973). Using our method in conservatively treated cases, the subjective results were acceptable in 49.1%, the functional results in 60.0% and the clinical results in 52.7%. In cases treated by operation the equivalent figures were 57.9%, 73.7% and 52.6%. The poorest results followed displaced medial condylar and medially tilted bicondylar fractures. Varus alignment of the tibial plateau was tolerated worse than valgus alignment.


Fracture of the tibial spine in adults and children. A review of 31 cases. We reviewed 19 adults and 12 children who had been treated for avulsion fractures of the tibial spine. Adult injuries have not previously been reported at length; most were caused by road-traffic accidents, and 68% were associated with other injuries, of which 58% were around the knee. The higher incidence of associated injuries in adults as compared with children, indicates that the injury is the result of greater energy and perhaps a different mechanism. The worse outcome in some adults was due to other associated intra-articular fractures and tears of the medial collateral ligament. Arthroscopy is useful in both diagnosis and treatment. Early accurate diagnosis and the correct treatment produce a good outcome.


Absorbable pins of self-reinforced poly-L-lactic acid for fixation of fractures and osteotomies. We reviewed 27 patients with small-fragment fractures or osteotomies treated by internal fixation with absorbable self-reinforced poly-L-lactide pins. The follow-up time ranged from eight to 37 months. The two most common indications were chevron osteotomy of the first metatarsal bone for hallux valgus and displaced fracture of the radial head. No redisplacements occurred, and there were no signs of inflammatory foreign-body reaction. Biopsy in two patients 20 and 37 months after implantation showed that no polymeric material remained.


Bone regrowth after surgical decompression for lumbar spinal stenosis. We reviewed 40 patients treated surgically for lumbar stenosis at an average time of 8.6 years after operation. In 32, total laminectomy had been performed and in eight bilateral laminotomy, both at one or more levels. Of the 16 patients with degenerative spondylolisthesis, ten had had a concomitant spinal fusion. Patients were assigned to one of four groups according to the amount of bone regrowth: group 0 had no regrowth and groups I, II, and III, had mild, moderate or marked regrowth, respectively. Only 12% of the patients showed no bone regrowth; 48% were assigned to group I, 28% to group II and 12% to group III. Imaging studies showed varying degrees of recurrent stenosis in patients with moderate or marked bone regrowth. All patients with degenerative spondylolisthesis showed bone regrowth, which was more severe in those who had not had a fusion. The clinical results were satisfactory in most of the patients with mild or no bone regrowth and significantly less good in those with moderate or marked regrowth. In the group with degenerative spondylolisthesis, the proportion of satisfactory results was significantly higher in patients who had had spinal fusion. The long-term results of surgery for lumbar stenosis depend both upon the amount of bone regrowth and the degree of postoperative vertebral stability.


Spinal cord monitoring during operative correction of neuromuscular scoliosis. We report our experience of the monitoring of spinal somatosensory evoked potentials in 60 patients with neuromuscular scoliosis. In 15 cases a significant change occurred in the trace when a sublaminar wire was tightened. There were no postoperative neurological deficits attributable to the surgery.


Upper-limb surgery for tetraplegia.

Upper-limb surgery for tetraplegia. We reviewed the results of reconstruction of 97 upper limbs in a consecutive series of 57 tetraplegic patients, treated from 1982 to 1990. Of these, 49 had functional and eight had cosmetic reconstructions. The principal functional objectives were to provide active elbow extension, hook grip, and key pinch. Elbow extension was provided in 34 limbs, using deltoid-to-triceps transfer. Hook grip was provided in 58 limbs, mostly using extensor carpi radialis longus to flexor pollicis longus transfer, and key pinch in 68, mostly using brachioradialis to flexor pollicis longus transfer. Many other procedures were employed. At an average follow-up of 37 months, 70% had good or excellent subjective results, and objective measurements of function compared favourably with other series. Revisions were required for 11 active transfers and three tenodeses, while complications included rupture of anastomoses and problems with thumb interphalangeal joint stabilisation and wound healing. We report a reliable clinical method for differentiating between the activity of extensor carpi radialis longus and brevis and describe a successful new split flexor pollicis longus tenodesis for stabilising the thumb interphalangeal joint. Bilateral simultaneous surgery gave generally better results than did unilateral surgery.


Osteoplastic repair of the atlas.

Osteoplastic repair of the atlas. Fractures of the atlas constitute 4% to 12% of all bony injuries of the cervical spine; most are treated successfully by a cervical orthosis. Nonunion may be associated with neck or scalp pain on movement and is treated conventionally by some form of craniocervical fusion, which restricts head movement. The authors describe a case in which direct repair of the bony ring with a titanium plate and screws allowed bone healing, relieved the symptoms and maintained a full range of neck movements. The titanium plate interfered little with postoperative MR and CT imaging.


A significant proportion of patients with osteosarcoma may belong to Li-Fraumeni cancer families. We studied the pedigrees of 17 index patients with osteosarcoma, recording malignant disease and cause of death for first- and second-degree relatives. There were seven cancers and five cancer deaths per 2151.5 person-years in first-degree relatives of osteosarcoma patients under the age of 50 years, a significantly greater incidence than in an age- and sex-matched population group (p < 0.001). This excess of malignancy was largely due to two families which fulfilled the criteria for the Li-Fraumeni cancer family syndrome. Both of these families were shown to have the genetic alterations in the p53 gene which have been implicated in this syndrome. Our study suggests that orthopaedic surgeons seeing new cases of osteosarcoma should arrange screening for familial malignancy.


Massive femoral allografts followed for 22 to 36 years. Report of six cases. Six massive femoral allografts followed up for 22 to 36 years are described. Three were intercalary, two were osteoarticular and one was a total femoral replacement. Their functional rating according to the Musculoskeletal Tumor Society System (Enneking 1987) averaged 82% (56 to 100). The radiographic score averaged 75% (48 to 100). Four allografts had suffered fractures, but three of these had later united to give good final scores. Our study shows that massive femoral allografts can function well for as long as 36 years.


Management and outcome in tibial dysplasia. Tibial dysplasia is a rare congenital deformity which must be distinguished from the more common fibular dysplasia. We have reviewed 24 patients with 35 affected legs. The classification system of Kalamchi and Dawe (1985) was found to be preferable to that of Jones, Barnes and Lloyd-Roberts (1978) as a guide to prognosis and management. We discuss the outcome of surgical treatment, recommending selective amputation for most cases.


Modification of the L'Episcopo procedure for brachial plexus birth palsies. We reviewed 19 children who had undergone a new modification of the L'Episcopo procedure for obstetric brachial plexus palsy. Through an axillary approach the latissimus dorsi tendon was re-routed anteriorly to the humerus and then anastomosed to the teres major tendon routed posteriorly. At an average follow-up of four years two months, the mean increase in shoulder abduction was 26 degrees and the mean increase in external rotation was 29 degrees. No neurovascular injury or postoperative infection occurred. Two patients had complications, and five did not gain from the procedure. The modified operation was relatively easier to perform and provided excellent cosmesis.


Ehlers-Danlos syndrome with soft-tissue contractures. We report four patients with a form of Ehlers-Danlos syndrome associated with soft-tissue contractures from birth and skin hyperalgesia. In early infancy, these cases were thought to be forms of arthrogryposis multiplex congenita, Larsen's syndrome or Marfan's syndrome. The characteristic triad of signs of Ehlers-Danlos disease gradually appeared from four to six years of age, allowing us to establish the correct diagnosis. We discuss the differential diagnosis of these connective-tissue disorders and the problems of the orthopaedic treatment of the associated joint deformities. Ehlers-Danlos syndrome is a heterogeneous group of metabolic diseases of collagen and our cases constitute a group which is distinct from the conventional types.


Osteomyelitis of the calcaneum.

Osteomyelitis of the calcaneum. We reviewed 52 cases of osteomyelitis of the calcaneum. The clinical symptoms and signs were well defined, but different and less dramatic than those of long-bone osteomyelitis. Blood cultures were positive in 41% of cases and tissue cultures in 91%. Routine haematological tests were of little value, and radiological changes were often delayed, and were absent in 12%. With early diagnosis, treatment with antibiotics alone was usually effective, but complications and chronic disease were more likely if there was delay. Early diagnosis is the key to successful treatment. We describe a new physical sign and consider that diagnosis is almost always possible by clinical methods.


Medial meniscus replacement by a tendon autograft. Experiments in sheep. In 20 skeletally mature female merino sheep, divided into four groups, we performed total medial meniscectomy, removal of the middle third of the patellar tendon, and tenotomy of the calcaneal tendon of the right hind leg. Group I (control) had no additional procedures. In the other three groups the medial meniscus was replaced by the middle third of the patellar tendon from the ipsilateral knee. The animals were killed at three (group II), six (group III), or 12 months (group IV) and the tendon-meniscus examined macroscopically, by light and scanning electron microscopy, and biomechanically. Remodelling of the tissue had taken place by 12 months but the failure stress and tensile modulus for the tendon-meniscus were lower than for the normal meniscus. Our evidence suggests that, in sheep, replacement of a meniscus by a tendon autograft may decrease the severity of the degenerative changes that occur after meniscectomy.


Repair of cervical nerve roots proximal to the root ganglia. An experimental study in sheep. An experimental model was established to investigate the possibility of repairing cervical nerve roots damaged above the dorsal root ganglion, as occurs in traction injuries of the brachial plexus. In four sheep the C6 root was divided and repaired within the dura using freeze-thawed muscle grafts. Recovery was assessed after eight months by electrophysiology and histology. Action potentials were recorded distal to the grafts in all four sheep, indicating regeneration of motor fibres. Histological examination showed regenerated fibres in the ventral roots below the grafts in all cases. These fibres could be traced distally to the brachial plexus. There was no evidence of recovery of dorsal roots.


Lipids and Dupuytren's disease.

Lipids and Dupuytren's disease. We studied prospectively the relationship between serum lipids and Dupuytren's disease of the hand in 85 patients, 65 men and 20 women. The Dupuytren patients had significantly higher fasting serum cholesterol and triglyceride levels than did the controls (p < 0.001). The raised levels of serum lipids appeared to be associated with the pathogenesis of Dupuytren's disease, and this may help to explain the high incidence of Dupuytren's disease in alcoholic, diabetic and epileptic patients, since these conditions are also associated with raised serum lipid levels.


Influence of coronary artery disease and left ventricular dysfunction on postoperative mortality in vascular surgery. In an attempt to assess the influence of coronary artery disease and left ventricular dysfunction on postoperative mortality, 107 patients consisting of 32 AAA resections and 44 aortoiliac and 31 femoropopliteal reconstructions were reviewed. All patients had a preoperative coronary angiograms and underwent cardiac catheterization to prevent cardiac-related deaths. Severe coronary artery disease was angiographically demonstrated in 40.6% of AAA, 31.8% of AI and 3.2% of FP patients. Severe left ventricular dysfunction was found in 21.9% of AAA, 18.2% of AI and 6.5% of FP. Both of the early and 5 (45.5%) of the 11 late deaths were caused by cardiac events. The nine-year survival rate (53.3%) in the patients with a documented combination of coronary artery disease and left ventricular dysfunction was significantly lower than those in patients with normal disease, normal left ventricular function and either documented coronary artery disease or left ventricular dysfunction. It is suggested from this series that left ventricular dysfunction is one of the most important risk factors in patients who undergo AAA resection and AI reconstruction and that severe disease and postoperative mortality are possibly reduced by certain kinds of interventional coronary therapies.


Influence of age on late results of valve replacement with porcine bioprostheses. From 1976 to 1987, 877 patients, aged 13 to 80 years, underwent valve replacement with a Carpentier-Edwards porcine bioprosthesis--330 aortic, 421 mitral, and 126 multiple replacements. Patients were divided into 4 age groups, according to their age at the time of surgery: group I, less than 45 years (190 patients), group II, 45 to 54 years (203 patients), group III, 55 to 64 years (304), and group IV, 65 to 80 years (180 patients). Follow-up was 97.5 complete, averaged 85 months, and totalled 5624 patient-years. There were 79 early deaths (9%), and 181 late deaths. Overall survival was 80% and 64% at 5 and 10 years. Operative mortality increased significantly with age, and late survival was significantly better for group I. Intrinsic structural deterioration was the most common cause for reoperation and was responsible for 91% of the 143 reoperations. Incidence of intrinsic structural deterioration and of reoperation decreased significantly with increasing age, with respective freedom rates at 10 years of 57 and 54 in group I compared to 93 and 92% in group IV. Freedom from treatment failure (including early deaths, valve-related deaths, and valve-related complications with permanent disability) was similar for all 4 groups ranging from 78 to 85%, without any correlation with age of the patient. At last follow-up, a similar proportion of patients of each group remained asymptomatic, patients in functional class I ranging from 50 to 55%. Thus, age is a major determinant of durability of bioprostheses, but it does not appear to have a significant role in the overall success of valve replacement.


Noninvasive assessment of internal thoracic artery for reoperative coronary artery surgery. To determine whether previous sternotomy alters internal thoracic artery (ITA) anatomy and flow characteristics, a duplex scanner was used for noninvasive evaluation of the ITA in 59 patients who were scheduled for reoperative coronary artery bypass surgery. The left ITA was insonated through the third intercostal space by use of a duplex scanner (5.0 MHz probe). Measurements of the ITA diameter (mm) and peak systolic velocity (cm/sec) were obtained; ITA flow was calculated from velocity and cross-sectional area. These findings were compared with the values obtained from 105 patients who were scheduled to undergo first-time (primary) coronary artery surgery during the same time period. In the reoperative group, preoperative mean ITA diameter was 2.26 +/- 0.06 mm; this was not significantly different from the primary group's mean ITA diameter of 2.15 +/- 0.04 mm (p = 0.09). Mean peak systolic velocity was 79.9 +/- 2.4 cm/sec and calculated systolic blood flow was 204.6 +/- 13.1 ml/min in the reoperative patients, as compared with 83.3 +/- 2.1 cm/sec and 189.5 +/- 8.6 ml/min in the primary group, respectively. Values were similar in both groups for the peak systolic velocity (p = 0.31) and calculated systolic blood flow (p = 0.32). These results suggest that previous heart surgery or sternotomy does not adversely affect ITA anatomy and flow characteristics. We conclude that ultrasonic imaging is an easily applicable technique for preoperative assessment of ITA in patients who have undergone previous sternotomy.


Simple hypothermic retrograde cerebral perfusion during aortic arch surgery. We have found that retrograde cerebral perfusion can be performed by simply elevating central venous pressure during aortic arch surgery. During hypothermia (15 degrees C) venous blood rich in oxygen perfuses the brain, while the lower half of the body is perfused with the descending aorta occluded. Fourteen cases of aortic arch aneurysm (11 males, 3 females) were treated by this method and evaluated. Median and lateral thoracotomies were performed in 6 and 8 cases, respectively. Cerebral circulatory arrest time was 65 +/- 14 min (32-93 min), and significant oxygen and lactate extraction were noted in the brain. Eleven cases showed no neurologic deficit, but loss of consciousness due to other causes occurred in 3. In conclusion, this method maintained the aerobic metabolism of the brain and protected it during prolonged cerebral circulatory arrest, simplifying the procedure and permitting both median and lateral approaches.


Retroperfusion and balloon support to improve coronary revascularization. Coronary venous retroperfusion and Intra-Aortic Balloon Pump (IABP) support are methods currently utilized to reduce ischemic damage prior to revascularization of acutely ischemic myocardium. This study was undertaken to determine whether combining coronary venous retroperfusion using Pressure Controlled Intermittent Coronary Sinus Occlusion (PICSO) with the IABP would result in improved salvage of ischemic myocardium. In 40 adult pigs, the second and third diagonal vessels were occluded with snares for 1 1/2 hours followed by 1/2 hours of cardioplegic arrest and 3 hours of reperfusion with the snares released. During the period of coronary occlusion prior to arrest, 10 pigs received the IABP, 10 had PICSO, 10 had PICSO+IABP, while 10 had no intervention (Unmodified). Ischemic damage was assessed by echocardiographic wall motion scores, myocardial pH, and the area of necrosis/area of risk using histochemical staining. Both PICSO and the IABP alone significantly reduced ischemic damage. However, the best wall motion scores, highest pH, and least necrosis was seen in the IABP+PICSO group. We conclude that the combination of coronary venous retroperfusion using PICSO and the IABP results in the most optimal recovery of acutely ischemic myocardium during emergent surgical revascularization.


Results of long-term venacavography study after placement of a Greenfield vena caval filter. Digital subtraction venacavography (DSV) as a follow-up examination was performed in 46 patients after placement of a standard stainless steel Greenfield vena caval filter (SGF). DSV is an exact method of visualizing the lumen of the inferior vena cava as well as intraluminal or captured thrombi. Eccentric filters with an angulation of more than 15 degrees were found in 8 patients causing a higher risk of pulmonary embolism from smaller emboli and propagating thrombi. Penetration of the caval wall by filter struts was however frequent (41%), without any damage to adjacent structures as demonstrated by CT scans. The long-term caval patency rate was 90%, with 4 cases of caval thrombosis. Two patients with an occluded inferior vena cava showed no venostasis. Although changes of the filter position usually remain asymptomatic, regular follow-up examinations seem necessary to recognize any impairment of the filter's clot trapping efficiency. Filters should only be used in patients with a risk of recurrent pulmonary embolism despite anticoagulation and patients with contraindications to anticoagulation therapy.


Haemolysis following mitral valve repair. We report the unusual occurrence of severe intra-vascular haemolysis following mitral valve repair. Mild to moderate mitral regurgitation was detected after repair, but severe haemolysis was the only indication for re-operation. Following prosthetic valve replacement there was an immediate cessation of haemolysis. We postulate that a small regurgitant jet directed against the teflon pledgets used in the repair was the reason for the haemolysis.


Internal mammary artery bypass: thirteen years of experience. Influence of angina and survival in 5125 patients. Internal mammary artery (IMA) bypass to the anterior descending coronary artery (ADA) was performed in 5125 patients from January 1978 to December 1990. The average age of patients was 68 years; males accounted for 68% (3485 patients) and 82% (4203) were NYHA Class III. Left ventricular function was impaired (ejection fraction < 40%) in 68% (3485 patients). The average number of additional saphenous vein graft (SVG) per patient was 2.2. Operative mortality was 1.8%. Mediastinitis occurred in 51 patients (1.0%). Reoperation for bleeding was necessary in 56 patients (1.1%). Perioperative myocardial infarction was seen in 102 patients (2.0%) and neurological complications occurred in 51 patients (1%). Repeat coronary angiography was performed in 1414 patients (28%) and demonstrated a patency rate of 96% in IMA grafts and 75% in SVG grafts (p < 0.001). Survival at 13 years was 80% from all causes and 90% when non-cardiac deaths were excluded. Recurrence of angina occurred in 768 patients (15%) and reoperation or PTCA was performed in 61 (1.2%). During the same time period, 2345 patients underwent coronary artery bypass utilizing solely SVG. Survival at 13 years was 68% from all causes and 78% when non-cardiac deaths were excluded (p < 0.001). Recurrent angina was present in 727 patients (31%) (< 0.001). This data suggests that long-term probability of cumulative survival and occlusion free survival were significantly greater and the probability of recurrent angina and reoperative CABG and death from cardiac causes were significantly less in the IMA patients and should be the conduit of choice in coronary bypass surgery.


Preservation of the pulmonary valve during intracardiac repair of tetralogy of Fallot. From January 1981 to December 1990, intracardiac repair of tetralogy of Fallot in 148 pediatric patients, with one surgical death, was directed toward preservation of the native pulmonary valve. Using the accepted preoperative angiographic criterion for the pulmonary valve annulus area (PVA) of 1.8 cm2/m2, 85 patients were candidates for transannular right ventricular outflow patch (TAP). However, in 54 patients with a mean PVA of 1.5 cm2/m2 (range 1.06-1.76), the valve was preserved without using TAP because the morphological changes (cusp thickening and annular distensibility) seemed acceptable for preservation in view of its probable hemodynamic efficiency and growth potential. A morphological classification of pulmonary valve changes has evolved. Retrospectively, 24 (77%) of the 31 patients with TAP had moderate to severe cusp thickening and ring rigidity; this incidence was significantly higher (p < 0.001) than that in preserved patients (18 of 54 or 33%). The incidence of morphological changes increased with operative age; that is, 2 of 13 (15%) patients younger than 1 year versus 23 of 40 (58%) patients older than 4 years (p < 0.01). All 54 patients with preserved pulmonary valves were catheterized one month postoperatively. The intraoperative right to left ventricular systolic pressure ratio (RVP/LVP) decreased significantly (p < 0.005) in one month, from a mean of 0.79 (range 0.44-1.36) to 0.57 (range 0.36-0.97). The PVA increased from a mean of 1.5 to 1.9 cm2/m2 (range 1.20-2.65), and the rate of its increase was significantly larger (p < 0.005) as operative age decreased. Pulmonary valve regurgitation of greater than mild degree occurred in 8 of 54 (15%) patients with the valve preserved.(ABSTRACT TRUNCATED AT 250 WORDS)


Effects of estrogen on vein grafts.

Effects of estrogen on vein grafts. The effect of estrogen on veins was evaluated in vitro and in vivo in three species. 17 beta-estradiol did not significantly alter 3H-thymidine uptake in vitro in segments from either canine femoral or human saphenous veins. In vivo in a rabbit carotid vein graft model, 17 beta-estradiol administration did not affect the development of intimal hyperplasia but was associated with a higher rate of graft thrombosis. These data suggest that the effects of estrogen on veins differs from the effects reported in arteries. These differences were seen both in vitro in veins taken from their normal location and in vivo in veins placed in the arterial circulation.


Endothelial cell injury in human saphenous veins following use of a circular valvulotome. With increasing use of nonreversed saphenous vein as a bypass conduit (either in-situ or translocated) several techniques of valve ablation are used. The present study was designed to assess the extent of endothelial damage caused by a circular blade valvulotome.


Extensive surgery for primary malignant lymphoma of the heart. The surgical treatment of primary malignant lymphoma of the heart in a 43-year-old man is reported. The tumor was extensively resected together with the right atrial wall and both venae cavae, and the resulting defect was repaired with a bovine pericardial patch. The postoperative course was fairly stable for more than 7 months following surgery, under a regular chemotherapy regimen. The patient died of acute pneumonia on the 234th postoperative day. The autopsy findings confirmed the diagnosis of primary malignant lymphoma of the heart.


Esmarch's bandage technique in distal bypass surgery. Esmarch's rubber bandage technique has been applied to 49 distal bypass surgeries in 46 patients during the past ten years. The primary and secondary patency rates at 5 years after femoro-tibial bypass surgery were 82% and 92%, respectively. This technique has the following advantages: (1) it minimizes surgical injury of the arterial wall because there is less dissection around the anastomotic site; (2) it decreases scar formation in the anastomotic area after surgery; (3) it maintains abundant muscular blood flow by preserving small branches to muscles, and (4) it provides a bloodless surgical field and easy handling for fine sutures without using vascular clamps. We consider that the avoidance of long circumferential dissection of the artery may play an important role in improving long-term patency in distal bypass surgery.


Do cytokines play a role in skeletal muscle ischemia and reperfusion? Cytokines, interleukin-1 (IL-1) and tumor necrosis factor (TNF) are known to mediate host cell response to sepsis, trauma, and myocardial ischemia. We have previously found increased levels of IL-1 in the venous effluent during the reperfusion phase of skeletal muscle ischemia in a canine model. This study was done to evaluate whether TNF also played a role in skeletal muscle ischemia-reperfusion injury since IL-1 and TNF have inter-related functions. In twelve adult mongrel dogs (28-32 kg) one gracilis muscle was subjected to six hours of normothermic ischemia followed by normothermic reperfusion. The contralateral side served as a control and remained normally perfused throughout the experiment. Gracilis venous samples were collected at pre-ischemia and one hour of reperfusion. Systemic (arterial) blood samples were taken simultaneously with the venous samples at one hour of reperfusion. At the end of the experiment the muscles were harvested and amount of necrosis quantitated by serial transections, nitroblue tetrazolium staining and computerized planimetry. Muscle necrosis on the experimental side was found to be 48.86 +/- 5.37%. Sera were analyzed for TNF activity using a bioassay. TNF levels in the gracilis venous effluent at one hour of reperfusion were not significantly different from the simultaneous systemic (arterial) levels (27.15 +/- 5.05 pg/ml vs 18.23 +/- 4.27 pg/ml). Pre-ischemic levels of TNF were 96.50 +/- 20.12 pg/ml, which was significantly higher than either venous or arterial levels obtained after one hour of reperfusion (p < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)


Temporal arteritis. Clinical implications for the vascular surgeon. Temporal arteritis is a systemic disease with local temporal artery symptoms, generalized constitutional symptoms and ocular involvement which affects the elderly. A study was undertaken to assess the clinical features of patients with temporal arteritis in a large multispecialty clinic practice. The study group consisted of 516 patients with clinical suspicion of temporal arteritis, of which 97 (18.8%) had a positive biopsy for arteritis. The records of these 74 females and 23 males were retrospectively reviewed for clinical implications of the disease. The average age of the cohort was 71.7 years, and male to female ratio was 1:3.2. There were 95 caucasians and 2 blacks. The most common clinical findings at presentation were abnormal temporal artery (65.9%), headache (64.8%), myalgias or arthralgias (46.6%), visual symptoms (37.1%) and fever (35.1%). Multiple symptoms were present in 97% of the patients. The erythrocyte sedimentation rate was > 50 mm per hour in 91% of patients. Corticosteroids were used to treat 95/97 patients. Twenty-seven (28%) of the patients completed treatment over an average 36.3 months. Sixty-eight (72%) other patients were either lost to follow-up, died, or continue on therapy. Complications of corticosteroid treatment occurred in 43 (44.3%) of patients, and complications of temporal arteritis occurred in 14 (14.4%). A review of biopsy data showed no difference in length of biopsy or yield of biopsy in the patients with positive and the patients with negative histology. Temporal arteritis is a systemic disease which responds well to corticosteroid treatment. Complications of the disease as well as of treatment make definitive diagnosis imperative.


Assembly and localization of the U1-specific snRNP C protein in the amphibian oocyte. To study the intranuclear localization of the U1-specific snRNP C protein and its assembly into U1 snRNPs, we injected transcripts encoding a myc-tagged C protein into amphibian oocytes. The distribution of protein translated from the injected RNA was essentially the same in continuous and pulse-label experiments. In both cases the C protein localized within the germinal vesicle in those structures known to contain U1 snRNPs, namely the lampbrush chromosome loops and hundreds of extrachromosomal granules called snurposomes. Oocytes were also injected with an antisense oligodeoxynucleotide that caused truncation of U1 snRNA at the 5' end. In these oocytes, myc-tagged C protein localized normally in the germinal vesicle and could be immunoprecipitated together with truncated U1 snRNA. These experiments suggest that the C protein can enter the germinal vesicle on its own and there associate with previously assembled U1 snRNPs. In transfected tissue culture cells, the myc-tagged C protein localized within the nucleus in a speckled pattern similar to that of endogenous U1 snRNPs.


Restricted distribution of mRNA produced from a single nucleus in hybrid myotubes. Although the proteins encoded by a single nucleus in multinucleated myotubes have a wide range of distributions within the myofiber, little is known about the distributions of their mRNAs. We have used hybrid myotubes in which one or a few nuclei are derived from myoblasts that express nonmuscle proteins to investigate this question. We find that three different mRNAs, encoding proteins that are, respectively, nuclear, cytoplasmic, and targeted to the ER, have similar distributions within myotubes. Each is confined to an area within approximately 100 microns of the nucleus that expresses it.


Concomitant subclavian and carotid artery disease: the need for a combined surgical correction. To determine the importance of carotid artery disease in patients undergoing revascularization of the proximal subclavian artery for a subclavian steal syndrome, an 18-year experience of 55 patients was reviewed. Concomitant carotid artery disease (> 50% stenosis) was present in 35 patients (Group I: 63.6%). Twenty patients (Group II: 36.4%) had no evidence of hemodynamically significant carotid disease. Twenty-five patients in Group I (Group IA: 71.4%) were treated by endarterectomy (CEA) for all their carotid lesions while one or both carotid lesions were left untreated in 10 patients (Group IB: 28.6%). The actuarial 5-year freedom rate from neurological events was 87.2% in Group IA, 34.9% in Group IB (p < 0.001) and 100% in Group II (Group IB vs. II, p < 0.001; Group IA vs. Group II, p = ns). All untreated carotid lesions had a deleterious effect on the early and late functional results after surgical reconstruction of the subclavian artery. We conclude that the combined correction of subclavian and carotid lesions should be recommended in every case.


Hepatic artery aneurysm simulating a pancreatic mass. A 61-year-old man presented with a mass in the right upper quadrant of the abdomen. Computer tomography scanning (CT) had initially suggested that the location was in the head of the pancreas. Further examination with ultrasonography and celiac angiography documented that the mass was an aneurysm of the common hepatic artery. The successful approach to its surgical management is described.


Isolated popliteal artery occlusion in the young. Three young patients with an isolated popliteal artery occlusion are presented, two with severe claudication and the third with a critical ischaemic foot. The work-up of these patients leads to certain aetiologic possibilities: microtrauma, smoking and contraceptive pills. Two of our patients underwent thromboembolectomy, the third managed conservatively. The follow-up was between six months and seven years and up to now all three patients remain well.


False aneurysms after aortic operations. Between 1980 and August 1991, we encountered 11 non-infected aortic pseudoaneurysms after aortic surgery. The interval between the initial operation and revision varied from 1.8 to 26.8 years (mean 15.2 years). Three were found more than 20 years after operation. Pulling off of and degrading of the silk sutures were considered to be the causes. All patients underwent operative correction without mortality. In thoracic aortic pseudoaneurysm repair, a temporary bypass between the right axillary artery and left external iliac artery was generally adopted. There was no need for heparinization with this technique.


Response of arterial smooth muscle cells to laminar flow. This study was designed to determine whether laminar flow influences the proliferation rate and morphology of smooth muscle cells (SMC) in culture. Bovine aortic SMC were subjected to a shear stress of 6 dyne/cm2. The control group was subjected to similar incubation conditions without flow. Flow cytometry demonstrated decreased proliferation rate in SMC subjected to laminar flow. This phenomenon was still evident 24 hours after flow cessation. SMC were examined by light and electron microscopy. SMC subjected to laminar flow aligned along the direction of flow assuming a spheric morphology. These changes were reversible after a 48-hour resting period. The degree of organization of actin, tubulin and other microfilaments was evaluated by indirect immunofluorescence. SMC subjected to shear stress showed a clear reorganization of the cytoskeleton with expression of stress fibres. These changes were reversible after a 48-hour resting period. These findings may contribute to understanding the mechanisms by which SMC in vivo respond to forces generated by blood flowing under pressure.


Multiple GTP-binding proteins regulate vesicular transport from the ER to Golgi membranes. Using indirect immunofluorescence we have examined the effects of reagents which inhibit the function of ras-related rab small GTP-binding proteins and heterotrimeric G alpha beta gamma proteins in ER to Golgi transport. Export from the ER was inhibited by an antibody towards rab1B and an NH2-terminal peptide which inhibits ARF function (Balch, W. E., R. A. Kahn, and R. Schwaninger. 1992. J. Biol. Chem. 267:13053-13061), suggesting that both of these small GTP-binding proteins are essential for the transport vesicle formation. Export from the ER was also potently inhibited by mastoparan, a peptide which mimics G protein binding regions of seven transmembrane spanning receptors activating and uncoupling heterotrimeric G proteins from their cognate receptors. Consistent with this result, purified beta gamma subunits inhibited the export of VSV-G from the ER suggesting an initial event in transport vesicle assembly was regulated by a heterotrimeric G protein. In contrast, incubation in the presence of GTP gamma S or AIF(3-5) resulted in the accumulation of transported protein in different populations of punctate pre-Golgi intermediates distributed throughout the cytoplasm of the cell. Finally, a peptide which is believed to antagonize the interaction of rab proteins with putative downstream effector molecules inhibited transport at a later step preceding delivery to the cis Golgi compartment, similar to the site of accumulation of transported protein in the absence of NSF or calcium (Plutner, H., H. W. Davidson, J. Saraste, and W. E. Balch. 1992. J. Cell Biol. 119:1097-1116). These results are consistent with the hypothesis that multiple GTP-binding proteins including a heterotrimeric G protein(s), ARF and rab1 differentially regulate steps in the transport of protein between early compartments of the secretory pathway. The concept that G protein-coupled receptors gate the export of protein from the ER is discussed.


Morphological analysis of protein transport from the ER to Golgi membranes in digitonin-permeabilized cells: role of the P58 containing compartment. The glycoside digitonin was used to selectively permeabilize the plasma membrane exposing functionally and morphologically intact ER and Golgi compartments. Permeabilized cells efficiently transported vesicular stomatitis virus glycoprotein (VSV-G) through sealed, membrane-bound compartments in an ATP and cytosol dependent fashion. Transport was vectorial. VSV-G protein was first transported to punctate structures which colocalized with p58 (a putative marker for peripheral punctate pre-Golgi intermediates and the cis-Golgi network) before delivery to the medial Golgi compartments containing alpha-1,2-mannosidase II and processing of VSV-G to endoglycosidase H resistant forms. Exit from the ER was inhibited by an antibody recognizing the carboxyl-terminus of VSV-G. In contrast, VSV-G protein colocalized with p58 in the absence of Ca2+ or the presence of an antibody which inhibits the transport component NSF (SEC18). These studies demonstrate that digitonin permeabilized cells can be used to efficiently reconstitute the early secretory pathway in vitro, allowing a direct comparison of the morphological and biochemical events involved in vesicular tafficking, and identifying a key role for the p58 containing compartment in ER to Golgi transport.


Evidence for a novel route of wheat storage proteins to vacuoles. Wheat seed storage proteins are deposited in protein bodies (PB) inside vacuoles, but their subcellular site of aggregation and their route to vacuoles are still controversial. In the present work, an ultra structural analysis of developing wheat endosperm at early to mid maturation was performed to address these issues. Golgi complexes were rarely detected, indicating that their role in wheat storage protein transport is limited. In contrast, a considerable amount of PB was detected in the cytoplasm. Many of these PB were surrounded by RER membranes and were enlarged by fusion of smaller PB. Small, electron lucent vesicles were detected around the surfaces of the PB in the cytoplasm, or attached to them, suggesting that such attachments and subsequent fusion of the vesicles with each other lead to the formation of small vacuoles containing PB inclusions. Immunogold labeling with serum raised against yeast-BiP, an ER-localized protein, demonstrated that the wheat BiP homolog was present within the PB in the cytoplasm as well as inside vacuoles. This confirmed that the PB were formed within the RER and that the Golgi complex was not involved in their transport to vacuoles. It is concluded that a considerable part of the wheat storage proteins aggregate into PB within the RER and are then transported as intact PB to the vacuoles by a novel route that does not utilize the Golgi complex.


Glycosome assembly in trypanosomes: variations in the acceptable degeneracy of a COOH-terminal microbody targeting signal. Trypanosomes compartmentalize most of their glycolytic enzymes in a peroxisome-like microbody, the glycosome. The specificity of glycosomal targeting was examined by expression of chloramphenicol acetyltransferase fusion proteins in trypanosomes and monkey cells. Compartmentalization was assessed by cell fractionation, differential detergent permeabilization, and immunofluorescence. The targeting signal of trypanosome phosphoglycerate kinase resides in the COOH-terminal hexapeptide, NRWSSL; a basic amino acid is not required. The minimal targeting signal is, as for mammalian cells, a COOH-terminal tripeptide related to -SKL. However, the acceptable degeneracy of the signal for glycosomal targeting in trypanosomes is considerably greater than that for peroxisomal targeting in mammals, with particularly relaxed requirements in the penultimate position.


Effects of null mutations and overexpression of capping protein on morphogenesis, actin distribution and polarized secretion in yeast. CAP1, the gene encoding the alpha subunit of Saccharomyces cerevisiae capping protein, was cloned using a probe prepared by PCR with primers based on the amino acid sequence of purified alpha subunit peptides. The sequence is similar to that of capping protein alpha subunits of other species but not to that of the S. cerevisiae capping protein beta subunit or any other protein. Null mutants of capping protein, prepared by deletion of the coding region of CAP1 and CAP2 separately or together, are viable and have a similar phenotype. Deletion of the gene for one subunit leads to a loss of protein for the other subunit. The null mutant has a severe deficit of actin cables and an increased number of actin spots in the mother. Cells are round and relatively large. These features are heterogeneous within a population of cells and vary with genetic background. Overexpression of CAP1 and CAP2 also causes loss of actin cables and cell enlargement, as well as the additional traits of aberrant morphogenesis and cell wall thickening. Capping protein null strains and overexpression strains exhibited normal polarized secretion during bud growth as demonstrated by labeling with fluoresceinated Con A. Projection formation and chitin deposition in response to mating pheromone, mating efficiency, and bud site selection were also normal in capping protein null strains. In addition, bulk secretion of invertase was unimpaired. These data indicate that actin cables are not required for polarized secretion in S. cerevisiae.


Cytosol- and clathrin-dependent stimulation of endocytosis in vitro by purified adaptors. Using stage-specific assays for receptor-mediated endocytosis of transferrin (Tfn) into perforated A431 cells we show that purified adaptors stimulate coated pit assembly and ligand sequestration into deeply invaginated coated pits. Late events in endocytosis involving membrane fission and coated vesicle budding which lead to the internalization of Tfn are unaffected. AP2, plasma membrane adaptors, are active at physiological concentrations, whereas AP1, Golgi adaptors, are inactive. Adaptor-dependent stimulation of Tfn sequestration requires cytosolic clathrin, but is unaffected by clathrin purified from coated vesicles suggesting that soluble and assembled clathrin pools are functionally distinct. In addition to adaptors and cytosolic clathrin other, as yet unidentified, cytosolic factors are also required for efficient coated pit invagination. These results provide new insight into the mechanisms and regulation of coated pit assembly and invagination.


5'nucleotidase is sorted to the apical domain of hepatocytes via an indirect route. In hepatocytes, all newly synthesized plasma membrane (PM) proteins so far studied arrive first at the basolateral domain; apically destined proteins are subsequently endocytosed and sorted to the apical domain via transcytosis. A mechanism for the sorting of newly synthesized glycophosphatidylinositol (GPI)-linked proteins has been proposed whereby they associate in lipid microdomains in the trans-Golgi network and then arrive at the apical domain directly. Such a mechanism poses a potential exception to the hepatocyte rule. We have used pulse-chase techniques in conjunction with subcellular fractionation to compare the trafficking of 5' nucleotidase (5NT), an endogenous GPI-anchored protein of hepatocytes, with two transmembrane proteins. Using a one-step fractionation technique to separate a highly enriched fraction of Golgi-derived membranes from ER and PM, we find that both 5NT and the polymeric IgA receptor (pIgAR) traverse the ER and Golgi apparatus with high efficiency. Using a method that resolves PM vesicles derived from the apical and basolateral domains, we find that 5NT first appears at the basolateral domain as early as 30 min of chase. However the subsequent redistribution to the apical domain requires > 3.5 h of chase to reach steady state. This rate of transcytosis is much slower than that observed for dipeptidylpeptidase IV, an apical protein anchored via a single transmembrane domain. We propose that the slow rate of transcytosis is related to the fact that GPI-linked proteins are excluded from clathrin-coated pits/vesicles, and instead must be endocytosed via a slower nonclathrin pathway.