Gly369Cys mutation in mouse FGFR3 causes achondroplasia by affecting both chondrogenesis and osteogenesis.

Missense mutations in fibroblast growth factor receptor 3 (FGFR3) result in several human skeletal dysplasias, including the most common form of dwarfism, achondroplasia. Here we show that a glycine-to-cysteine substitution at position 375 (Gly375Cys) in human FGFR3 causes ligand-independent dimerization and phosphorylation of FGFR3 and that the equivalent substitution at position 369 (Gly369Cys) in mouse FGFR3 causes dwarfism with features mimicking human achondroplasia. Accordingly, homozygous mice were more severely affected than heterozygotes. The resulting mutant mice exhibited macrocephaly and shortened limbs due to retarded endochondral bone growth and premature closure of cranial base synchondroses. Compared with their wild-type littermates, mutant mice growth plates shared an expanded resting zone and narrowed proliferating and hypertrophic zones, which is correlated with the activation of Stat proteins and upregulation of cell-cycle inhibitors. Reduced bone density is accompanied by increased activity of osteoclasts and upregulation of genes that are related to osteoblast differentiation, including osteopontin, osteonectin, and osteocalcin. These data reveal an essential role for FGF/FGFR3 signals in both chondrogenesis and osteogenesis during endochondral ossification.

Expression of fibrinolytic genes in atherosclerotic abdominal aortic aneurysm wall. A possible mechanism for aneurysm expansion.

Expansion of atherosclerotic abdominal aortic aneurysm (AAA) has been attributed to remodeling of the extracellular matrix by active proteolysis. We used in situ hybridization to analyze the expression of fibrinolytic genes in aneurysm wall from eight AAA patients. All specimens exhibited specific areas of inflammatory infiltrates with macrophage-like cells expressing urokinase-type plasminogen activator (u-PA) and tissue-type PA (t-PA) mRNA. Type 1 PA inhibitor (PAI-1) mRNA was expressed at the base of the necrotic atheroma of all specimens and also within some of the inflammatory infiltrates where it frequently colocalized in regions containing u-PA and t-PA mRNA expressing cells. However, in these areas, the cellular distribution of the transcripts for t-PA and u-PA extended far beyond the areas of PAI-1 expression. These observations suggest a local ongoing proteolytic process, one which is only partially counteracted by the more restricted expression of PAI-1 mRNA. An abundance of capillaries was also obvious in all inflammatory infiltrates and may reflect local angiogenesis in response to active pericellular fibrinolysis. The increased fibrinolytic capacity in AAA wall may promote angiogenesis and contribute to local proteolytic degradation of the aortic wall leading to physical weakening and active expansion of the aneurysm.

The transmembrane mutation G380R in fibroblast growth factor receptor 3 uncouples ligand-mediated receptor activation from down-regulation.

A point mutation, Gly380Arg, in the transmembrane domain of fibroblast growth factor receptor 3 (FGFR3) leads to achondroplasia, the most common form of genetic dwarfism in humans. This substitution was suggested to enhance mutant receptor dimerization, leading to constitutive, ligand-independent activation. We found that dimerization and activation of the G380R mutant receptor are predominantly ligand dependent. However, using both transient and stable transfections, we found significant overexpression only of the mutant receptor protein. Metabolic pulse-chase experiments, cell surface labeling, and kinetics of uptake of radiolabeled ligand demonstrated a selective delay in the down-regulation of the mutant receptor. Moreover, this receptor was now resistant to ligand-mediated internalization, even at saturating ligand concentrations. Finally, transgenic mice expressing the human G380R mutant receptor under the mouse receptor transcriptional control demonstrated a markedly expanded area of FGFR3 immunoreactivity within their epiphyseal growth plates, compatible with an in vivo defect in receptor down-regulation. We propose that the achondroplasia mutation G380R uncouples ligand-mediated receptor activation from down-regulation at a site where the levels and kinetics of FGFR3 signals are crucial for chondrocyte maturation and bone formation.

Close similarity among streptavidin-like, biotin-binding proteins from Streptomyces.

Two strains of Streptomyces venezuelae were found to produce high-affinity, biotin-binding proteins, termed streptavidin v1 and v2, respectively. Both proteins were isolated to purity, and their corresponding genes were cloned and sequenced. Compared to streptavidin from S. avidinii, streptavidin v1 had only a single amino acid substitution and streptavidin v2 showed 9 such differences. The substitutions were remarkably conservative, none of which affected the amino acid residues known to be important to the biotin-binding properties or to the structure of the tetrameric protein. The results also indicate that the biosynthesis of such biotin-binding proteins is not simply a curious anomaly in a single species of Streptomyces. It is suggested that the classification of S. avidinii as a unique species should be reconsidered. The occurrence of these proteins appears to be linked to the production of an unusual synergistic antibiotic complex.

Vascular lesions associated with impotence in diabetic and nondiabetic arterial occlusive disease.

Translumbar aortographies performed in 91 patients for limiting leg ischemia were reviewed, and stenosis was graded by points from one (normal vessel) to five (complete occlusion) for each vessel. Of 62 nondiabetic patients, 18 (29 per cent) were impotent, while of 29 diabetics, 17 (58.6 per cent) were impotent (p less than 0.01). Significantly greater stenosis (p less than 0.005) was found in the internal pudendal arteries of impotent patients when compared statistically with potent patients. This was true for the group as a whole, for diabetics and nondiabetics, and for patients over 50 years old both with and without diabetes. There was no significant difference in the extent of stenosis of the iliac arteries (common and internal) between potent and impotent patients. There was also no significant difference in the pattern of stenosis between diabetic and nondiabetic patients in the group as a whole and also in the potent and impotent subgroups analyzed separately. Neither diminished femoral pulses nor aortographic evidence of external iliac and common femoral arterial stenosis correlated significantly with impotence. These observations indicate that vascular lesions are as important in diabetics as in nondiabetics in the genesis of impotence. Clinical implications regarding diagnostic investigations and treatment are discussed.

Structural features of the combining site region of Erythrina corallodendron lectin: role of tryptophan 135.

The role of Trp 135 and Tyr 108 in the combining site of Erythrina corallodendron lectin (ECorL) was investigated by physicochemical characterization of mutants obtained by site-directed mutagenesis, hemagglutination-inhibition studies, and molecular modeling, including dynamics simulations. The findings demonstrate that Trp 135 in ECorL: (1) is required for the tight binding of Ca2+ and Mn2+ to the lectin because mutation of this residue into alanine results in loss of these ions upon dialysis and concomitant reversible inactivation of the mutant; (2) contributes to the high affinity of methyl alpha-N-dansylgalactosaminide (MealphaGalNDns) to the lectin; and (3) is solely responsible for the fluorescence energy transfer between the aromatic residues of the lectin and the dansyl group in the ECorL-MealphaGalNDns complex. Docking of MealphaGalNDns into the combining site of the lectin reveals that the dansyl moiety is parallel with the indole of Trp 135, as required for efficient fluorescence energy transfer, in one of the two possible conformations that this ligand assumes in the bound state. In the W135A mutant, which still binds MealphaGalNDns strongly, the dansyl group may partially insert itself into the place formerly occupied by Trp 135, a process that from dynamics simulations does not appear to be energetically favored unless the loop containing this residue assumes an open conformation. However, a small fraction of the W135A molecules must be able to bind MealphaGalNDns in order to explain the relatively high affinity, as compared to galactose, still remaining for this ligand. A model for the molecular events leading to inactivation of the W135A mutant upon demetallization is also presented in which the cis-trans isomerization of the Ala 88-Asp 89 peptide bond, observed in high-temperature dynamics simulations, appears not to be a required step.

Controlled trial of propranolol in intermittent claudication.

Seven patients (5 with arteriosclerosis obliterans and 2 with Buerger's disease) completed a two-phase double-blind crossover trial of propranolol in intermittent claudication. Performance was measured on a moving treadmill. In the initial phase, the patients were hospitalized in order to determine an "effective" dose of propranolol. Improvement was noted in all: after 1,600 mg in 5 and after 240 mg and 600 mg in the others. The controlled phase was carried out on an outpatient basis over 8 weeks, the patients receiving propranolol and placebo in a random manner, each for two 2-week periods. Comparison of matched periods of drug and placebo revealed no advantage for propranolol. Patients' performances deteriorated with time. None of the patients evidenced deterioration of occlusive peripheral arterial disease that could be attributed to propranolol, in spite of the high doses used.

The amino acid sequence of Erythrina corallodendron lectin and its homology with other legume lectins.

The primary sequence of Erythrina corallodendron lectin was deduced from analysis of the peptides derived from the lectin by digestion with trypsin, chymotrypsin, Staphylococcus aureus V8 protease, elastase and lysylendopeptidase-C, and of fragments generated by cleavage of the lectin with dilute formic acid in 6 M guanidine hydrochloride. Purification of the individual peptides was achieved by gel filtration, followed by reverse phase HPLC. The glycosylation site (Asn17-Leu18-Thr19) was deduced from analysis of the glycopeptide isolated from a pronase digest of the lectin before and after deglycosylation of the glycopeptide with endoglycosidase F. Comparison of the sequence of 244 residues thus obtained with those of 9 other legume lectins revealed extensive homologies, including 39 invariant positions and 60 partial identities. These data provide further evidence for the conservation of the lectin gene in leguminous plants.

Cloning, sequence analysis and expression in Escherichia coli of the cDNA encoding a precursor of peanut agglutinin.

The cDNA coding for pre-peanut agglutinin (PNA) was isolated from a bacterial expression library. It codes for a polypeptide of 273 amino acids composed of a hydrophobic signal peptide of 23 amino acids and a mature protein of 250 amino acids. The sequence of the latter is identical to that of native PNA, determined very recently by conventional methods, except that it contains 14 additional amino acids at the C-terminus. Bacterial cells harboring a plasmid with the prePNA-cDNA, produced two PNA cross-reacting proteins: one migrated on SDS-PAGE identically with the native lectin (apparent mol. wt. 31 kDa); the other, at 35 kDa, was a beta-galactosidase pre-PNA fusion protein. The former protein possessed an N-terminal sequence identical to that of the mature, native PNA, suggesting that it was processed from the 35 kDa prePNA precursor. Only the 31 kDa protein was exported into the bacterial periplasmic space, and had the ability to bind to galactose-Sepharose. The isolated processed protein had the same hemagglutinating activity as the native lectin, when assayed with sialidase-treated human erythrocytes. Like the native lectin, it did not agglutinate the untreated cells, was not inhibited by N-acetylgalactosamine, and was inhibited by Gal beta 1----3GalNAc 30-times more strongly than by galactose.

FGF receptors ubiquitylation: dependence on tyrosine kinase activity and role in downregulation.

A crucial aspect of ligand-mediated receptor activation and shut-down is receptor internalization and degradation. Here we compared the ubiquitylation of either wild type or a K508A 'kinase-dead' mutant of fibroblast growth factor receptor 3 (FGFR3) with that of its naturally occurring overactive mutants, G380R as in achondroplasia, or K650E involved in thanatophoric dysplasia. Fibroblast growth factor receptors ubiquitylation was found to be directly proportional to their intrinsic tyrosine kinase activity, both of which could be blocked using kinase inhibitors. Despite excessive ubiquitylation, both overactive mutants failed to be efficiently degraded, even when challenged with ligand or overexpression of c-Cbl, a putative E3 ligase. We conclude that phosphorylation is essential for FGFR3 ubiquitylation, but is not sufficient to induce downregulation of its internalization resistant mutants.

Modification by site-directed mutagenesis of the specificity of Erythrina corallodendron lectin for galactose derivatives with bulky substituents at C-2.

Examination of the three-dimensional structure of Erythrina corallodendron lectin (ECorL) in complex with a ligand (lactose), the first of its kind for a Gal/GalNAc-specific lectin [(1991) Science 254, 862-866], revealed the presence of a hydrophobic cavity, surrounded by Tyr108 and Pro134-Trp135, which can accommodate bulky substituents such as acetamido or dansylamido (NDns) at C-2 of the lectin-bound galactose. Comparison of the primary sequence of ECorL with that of soybean agglutinin, specific for galactose and its C-2 substituted derivatives, and of peanut agglutinin, specific for galactose only, showed that in soybean agglutinin, Tyr108 is retained, and Pro134-Trp135 is replaced by Ser-Trp, whereas in peanut agglutinin, the former residue is replaced by Thr and the dipeptide by Ser-Glu- Tyr-Asn. Three mutants of ECorL were therefore constructed: L2, in which Pro134-Trp135 was replaced by Ser-Glu-Tyr-Asn; Y108T, in which Tyr108 was replaced by Thr and the double mutant L2; Y108T. They were expressed in Escherichia coli, as done for recombinant ECorL [(1992) Eur. J. Biochem. 205, 575-581]. The mutants had the same hemagglutinating activity as native or rECorL. Their specificity for galactose, GalNAc and Me beta GalNDns was examined by inhibition of hemagglutination and of the binding of the lectin to immobilized asialofetuin; in addition, their association constants with Me alpha GalNDns and Me beta GalNDns were measured by spectrofluorimetric titration. The results showed that Y108T had essentially similar specificity as the native and recombinant lectins. The affinity of L2 and L2;Y108T for galactose was also the same as ECorL, but they had a lower affinity for GalNAc and markedly diminished affinity for the dansyl sugars (up to 43 times, or 2 kcal, less). This appears to be largely due to steric hindrance by the two additional amino acids present in the cavity region in these mutants. Our findings also provide an explanation for the inability of PNA to accommodate C-2-substituted galactose derivatives at its primary subsite.

Vascular insufficiency quantitatively aggravates diabetic neuropathy.

The effect of lower-limb ischemia on the severity of neuropathy was examined in 48 diabetic patients with peripheral vascular disease. The severity of the vascular disease, as determined by medical history, physical findings, and laboratory data, was scored for each leg. Neuropathy was rated clinically and based on the results of nerve conduction studies of the common peroneal, posterior tibial, and sural nerves. A significant correlation was found between the vascular scores and neurologic variables of the two legs, most strikingly so in electrophysiologic data, with coefficients of .6 to .7. Nondiabetic control patients showed no evidence of neuropathy, regardless of the severity of ischemia, whereas diabetic controls without limb ischemia showed symmetrical neuropathy. These findings support the hypoxic theory in the pathogenesis of diabetic neuropathy.

Gustatory phenomena after upper dorsal sympathectomy.

In a series of 100 bilateral upper dorsal sympathectomies performed for palmar hyperhidrosis, gustatory sweating and other gustatory phenomena were reported by 68 of 93 patients (73%), followed up for an average of 1 1/2 years. These gustatory phenomena were quite different from physiologic gustatory sweating: a wide range of gustatory stimuli caused a variety of phenomena in varied locations. There was a negative correlation between the incidence of these phenomena and the occurrence of Horner's syndrome after sympathectomy. Analysis of our observations, and of clinical and experimental work of others, leads to the conclusion that gustatory phenomena after upper dorsal sympathectomy are the result of preganglionic sympathetic regeneration or collateral sprouting with aberrant synapses in the superior cervical ganglion.

Pulmonary functional abnormalities after upper dorsal sympathectomy. A comparison between the supraclavicular and transaxillary approaches.

Results of pulmonary function studies were compared in two groups of 12 patients each, in whom upper dorsal sympethectomy was performed by the supraclavicular or by the transaxillary approach. Patients were evaluated clinically, radiologically and functionally before operation and again three weeks, three months and six months after denervation. Findings suggest that an increase in small airway resistance concomitant with some degree of pneumoconstriction occurred after upper dorsal sympathectomy by both routes. Musclar transection and possible phrenic nerve retraction damage due to the operative procedure could not be the cause of the above abnormalities because the inspiratory and expiratory forces, inspiratory peak flow and diaphragmatic movement were not significantly reduced after operation by both approaches. However, in a few cases, extrapleural hematomas, segmental atelectasis and relaxation of the daiphrgm could have contributed to the loss of the lung volume. This was evident only in the early period and was obvious in the transaxillary approach group.

Effect of dehydration and cardiac tamponade on superior mesenteric artery flow: role of vasoactive substances.

The role of serotonin, catecholamines, and angiotensin in the pathogenesis of mesenteric low flow states was investigated in anesthetized dogs by measurement of blood flows with electromagnetic flow meters. During dehydration or cardiac tamponade, a disproportionate decrease in superior mesenteric artery flow was demonstrated, compared with aortic flow, but renal artery flow was relatively better maintained. Depletion of serotonin and catecholamines by pretreatment with reserpine or blocking serotonin's action with methysergide did not alter the disproportionate reduction in mesenteric flow. Disproportionate superior mesenteric artery flow during dehydration and tamponade was associated with increased levels of circulating angiotensin and virtually was abolished by bilateral nephrectomy, by inhibition of enzymatic conversion of angiotensin I to II by Bothrops nonapeptide, and by competitive inhibition of angiotensin II with 1-sar, 8-ala angiotensin II. Exogenous angiotensin administered intravenously to dogs not protected by drug treatment disproportionately decreased superior mesenteric artery flow with less effect on renal artery flow. These results are compatible with the hypothesis that increased circulating levels of angiotensin during dehydration and tamponade contribute to the disproportionate reduction in superior mesenteric artery flow in the anesthetized dog.

Autoimmune mechanisms in thromboangiitis obliterans (Buerger's disease): the role of tobacco antigen and the major histocompatibility complex.

This study is a continuation of our previous work that showed that patients with thromboangiitis obliterans (TAO; Buerger's disease) demonstrate a cell-mediated immune response to human artery type-specific collagens. To investigate the role of cigarette smoking in patients with TAO, cellular and humoral sensitivity was tested to a tobacco glycoprotein (TGP) antigen in 13 patients with Buerger's disease, 16 healthy smokers, and 12 nonsmoking healthy young male subjects. In this study, patients with Buerger's disease and healthy smokers had the same rate of cellular response to TGP, whereas nonsmokers did not respond. All three groups had a 30% to 40% measurable antibody response to TGP. If TGP has an immunologic role in the pathogenesis of TAO, an additional factor (or factors) may be operative. A specific genetic makeup may be one such factor, although at this stage other pathogenic mechanisms cannot be ruled out. Eleven patients with Buerger's disease and two control groups of 10 young healthy smoking male subjects and 12 young nonsmokers underwent histocompatibility leukocyte antigen (HLA) typing. Patients with Buerger's disease had a statistically significantly higher frequency of HLA-DR4 and a significantly lower frequency of the HLA-DRW6 antigen than had both control groups. Because similar findings have been reported in other autoimmune diseases, this observation may serve as further evidence that an autoimmune mechanism is involved in Buerger's disease.

Left renal vein division in abdominal aortic aneurysm operations. Effect on renal function.

We examined the effect of left renal vein (LRV) division during abdominal aortic aneurysm operations on renal function during the recovery period. Fifteen patients with LRV division were compared with 26 patients in whom the LRV was not ligated. These two groups of patients did not differ significantly in any of their preoperative characteristics, operative management, or postoperative complications. Preoperative, highest postoperative, and predischarge levels of plasma urea and creatinine, as well as urinary sediment, were compared in both groups. Left renal vein division could not be implicated as a cause of renal function deterioration and was found to be a safe, useful adjunct to abdominal aortic aneurysm surgery.

Changes in plasmatic tissue-type plasminogen activator and plasminogen activator inhibitor activity during acute arterial occlusion associated with severe ischemia.

Severe lower limb ischemia in patients with acute arterial occlusion was associated with a significant increase in systemic fibrinolytic activity. Plasmatic level of t-PA activity was twice the normal value at the peak of ischemia. This level declined gradually within no less than 40 hours after reperfusion procedure or limb amputation had reverted the ischemic state. In spite of major tissue damage and surgical trauma, plasmatic PAI activity stayed within normal range, and did not increase within the first 24 hours postoperatively. These findings strongly suggest that acute ischemia initiates systemic induction of excessive and continuous release of t-PA, which outweighs any anticipated increase in PAI activity.

Hemorrhagic complications encountered on a surgical service. A 7 year survey.

An analysis of 205 episodes of bleeding encountered during a 7 year period by the staff of a surgical service has been described. Of 168 postoperative bleeding episodes, 80 were associated with anticoagulant or antiaggregant treatment, 51 were caused by technical error, and 37 were associated with sepsis. Of 18 hemorrhages after invasive procedures, 12 were associated with anticoagulants. In the whole series, in 33 of 111 episodes in patients receiving antithrombotic drugs, there was some deviation from standard accepted protocols. Bleeding occurred in the surgical wound or at the site of operation or puncture in 154 instances. Some of the bleeding episodes, especially at other sites, presented in an unusual manner and posed a diagnostic problem. Bleeding was responsible for death or contributed to death in 11 patients, and in 32 patients, it caused operation or reoperation. Other short- and long-term morbidity was also caused. As in many complications, prevention is of the essence.

Reoperation. Planned and unplanned.

Reoperation is an integral part of the damage control sequence. Each type of reoperation represents an entirely different operative profile with different tactical considerations. This article discusses in detail the key decisions and techniques of planned and unplanned reoperation.