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Advances in imaging have led to improved sensitivity in the diagnosis of diseases that involve the major salivary glands. Ultrasound (US), plain radiography and sialography, magnetic resonance imaging (MRI), computed tomography (CT), and nuclear scintigraphy/positron emission tomography (PET) all play a part, and imaging often assists in the planning of further management, operative or otherwise. We review the methods used for imaging the major salivary glands, and apply the indications for these methods to the principal pathological processes., (Copyright © 2010 The British Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.)

The successful use of live attenuated viral and bacterial vaccines depends not only on the proper choice and delivery of the microorganisms, but also on maintaining the sufficient potency required for an immune response. The inherent lability of live organisms presents a particular formulation challenge in terms of stabilizing and preserving vaccine viability during manufacturing, storage, and administration. This review examines pharmaceutical approaches to the stabilization, formulation, and lyophilization of biological macromolecules in general, as well as the specific applicability of these principles to live attenuated viral and bacterial vaccines. Several formulation development case studies with live vaccines are presented. In addition, comparative stability data are summarized for many other live viral and bacterial preparations. Various pharmaceutical issues with conventional and novel delivery systems for administration of parenteral and oral live vaccines are also discussed.

The N-methyl-D-aspartate (NMDA) subclass of glutamate receptors was examined in newborn infants dying between 25 weeks' gestation and term, either from acute cerebral hypoxia, or from other noncerebral conditions incompatible with life. Frontal, occipital, temporal, and motor cortex tissue samples were obtained at autopsy (post mortem delay: median, 45.9 hr; range, 24-96 hr) and frozen for subsequent [3H]MK801 homogenate binding assays. Whereas no significant variation was observed in ligand affinity (KD), in all cases receptor density (BMAX) increased with gestational age, in occipital cortex (27 weeks, BMAX = 222 +/- 44 fmol x mg protein(-1); 39 weeks, 439 +/- 42 fmol x mg protein[-1]), but not in motor or temporal cortex. The gestational-age increase also occurred in control frontal cortex (27 weeks, 284 +/- 80; 39 weeks, 567 +/- 40 fmol x mg protein[-1]), but was significantly less marked in frontal cortex in hypoxia cases (27 weeks, 226 +/- 90; 39 weeks, 326 +/- 47 fmol x mg protein[-1]). In all cortical areas except temporal, the maximal response to glutamate did not vary across case groups. Hypoxia cases showed an increased response to glutamate enhancement selectively in temporal cortex. Binding site density did not correlate with degree of hypoxia as assessed pathologically, suggesting that receptor differences preceded the hypoxic episode. Regional differences in glutamate-NMDA receptor sites may underlie increased vulnerability to hypoxia at birth.

A variety of biophysical techniques have been employed to examine the size and conformational integrity of highly purified hepatitis A virus (HAV) in solution (purified HAV particles are subsequently formalin-inactivated and adsorbed to aluminum salts for use as the vaccine VAQTA). The size of HAV particles was assessed by a combination of electron microscopy, sedimentation velocity, and dynamic light scattering. The effect of ionic strength and temperature on the overall conformational stability of HAV was determined by a combination of intrinsic HAV protein fluorescence, fluorescent probes of both RNA and protein, and UV-visible spectroscopy. A major structural change in HAV occurs near 60 degrees C with the addition of 0.2 M magnesium chloride enhancing the thermal stability of HAV by approximately 10 degrees C. Salt concentrations above 0.2 M, however, decrease the solubility of HAV. The effect of pH on the physical properties of HAV particles was monitored by dynamic light scattering, analytical size exclusion HPLC, and interaction with fluorescent dyes. HAV particles undergo a substantially reversible association/aggregation at pH values below 6 with the concomitant exposure of previously buried hydrophobic surfaces below pH 4. These results are in good agreement with previous studies of HAV thermal stability under extreme conditions in which the irreversible inactivation of the viral particles was measured primarily by the loss of viral infectivity. The wide variety of biophysical measurements described in this work, however, directly monitor structural changes as they occur, thus providing a molecular basis with which to monitor HAV stability during purification and storage.

Nine high-energy pilon fractures with severe soft tissue injuries were treated by a medial external fixator with an articulated ankle-hinge and limited internal fixation (1.7 screws per case). A 100% union rate was achieved; however, there was a 100% complication rate associated with the fixator. Both B3 fractures required a vertical transarticular pin to maintain reduction. Seven C2 fractures suffered calcaneal screw loosening and drainage, necessitating removal of the fixator prior to union. Due to these complications, the articulated ankle hinge could not be utilized. At a minimum of 6 months follow up, eight of nine fractures had acceptable radiographic and early clinical results.

Eighty patients with pelvic fractures, without hip, spine injuries with neurologic deficits, or traumatic cognitive deficits, 61% treated with external fixators, were reviewed with greater than 5 years of followup. The Short Form-36 General Health Survey, the Iowa pelvic scores, and additional questionnaires concerning return to previous sexual function, occupation, and recreation, and a perception of the worst sequelae of their pelvic trauma were administered. Fractures were classified according to Tile as 25 undisplaced Class A, 31 rotationally displaced Class B, and 24 vertically displaced Class C fractures. Injury severity scores, associated injuries, and mechanisms of injuries were similar across Tile classes. Statistical analysis compared Tile Classes A with B with C and Classes A with B and C. Variations by Tile class among the Short Form-36 survey, Iowa pelvic score, or questionnaire result's were not seen. Approximately 75% of patients returned to their previous sexual function and 80% to their previous occupation. Similarities comparing undisplaced with displaced pelvic fractures question whether reduction of pelvic ring injuries can alter patient long term functional outcomes.

Twenty-nine patients with tibial diaphyseal fractures had external fixators applied to treat their initial fractures and underwent nonreamed solid core nailing for nonunion (> 22 weeks). Fifteen Alta, 11 Lottes, and 3 Rush nails were used. The original fracture grades were: 1 closed, 1 grade I, 7 grade II, 3 grade IIIA, and 17 grade IIIB. The duration of nonunion was 51 weeks (average: 22 to 173). The average duration of external fixation was 19 weeks (range: 9 to 47). The average interval from fixator removal to nail was 32 weeks (range: 0 to 156). Twenty-eight patients were seen at > 12 months' follow up (average: 35; range: 10 to 58) and completion of treatment. Twenty-seven patients' fractures united at an average of 14 weeks (range: 6 to 40). One patient required a reamed exchange nailing at 39 weeks and united 58 weeks post-exchange nailing. One patient had a persistent asymptomatic nonunion. One patient developed a stress fracture 49 months post nail requiring an exchange nailing. Eleven fractures had a pre-nail deformity of 10 degrees or more; 9 were corrected. Eight patients had pre-nail wound and five had pre-nail pin tract infections. Except for two cases, all of these infections were treated successfully with surgical debridement and/or antibiotics prior to nail insertion. Two patients had their nail inserted through active infections that could not be controlled with an unstable fracture. One patient healed uneventfully in 6 weeks. One required two subsequent debridements. There were no other infections. The authors conclude that nonreamed solid core nailing is an acceptable treatment for tibial diaphyseal nonunions following external fixation.

The complexity of vaccines creates a unique formulation challenge. Vaccines may consist of one or more types of antigenic component including live attenuated or killed viral or bacterial particles, polysaccharides, proteins, polynucleotides and particle conjugates. In addition, other excipients such as adjuvants may be present. Not only must the chemical and structural integrity of the various components be maintained, but immunogenicity must be ensured. The inherent lability of vaccines can critically limit their distribution, administration, and efficacy in parts of world where it is difficult to maintain a cold chain. Combination with other vaccines and oral administration may also compromise vaccine stability. Successful vaccine stabilization strategies include both empirical efforts to screen and identify appropriate stabilizers and environmental conditions and more rational approaches toward developing an understanding of the causes and mechanisms of vaccine inactivation. In principle, by elucidating the conformational and chemical pathways of macromolecular inactivation, more rational strategies to minimize their occurrence can be adopted. This presentation will review the application of classical techniques such as viral plaque assays to identify vaccine stabilizers by empirical testing. The potential of using various biophysical techniques (both hydrodynamic and spectroscopic methods) to characterize the physicochemical stability of purified vaccine preparations (Hepatitis A and B) is also explored.

A retrospective comparison of dynamic and static locking mode nonreamed nails in 88 closed, Grades I and II open tibial fractures is presented. Amount of time and number of reoperations required to unite fractures were compared for dynamic (Group 1, n = 31) and static locked (Group 2, n = 13) Winquist I and II fractures, and dynamic (Group 3, n = 14) and static locked (Group 4, n = 30) Winquist III, IV, and segmental fractures. Total reoperations also were compared; Tibias treated with dynamic nails united in an average of 20 weeks, with 3 reoperations; tibias treated with static locked nails united in an average of 30 weeks, with 21 reoperations. Group 1 fractures united in an average of 20 weeks, with 1 reoperation; Group 2 fractures united in an average of 32 weeks, with 4 reoperations. Group 3 fractures united in an average of 20 weeks; Group 4 fractures united in an average of 29 weeks, with 11 reoperations. In the group of tibias treated with dynamic nails, 3 additional operations were done; in the group of tibias treated with static locked nails, 7 additional operations were done. There was 1 infection and 3 deformities. Static locking mode appeared to delay union, especially when Groups 1 and 2 are compared; these fractures can be nailed without locking screws.

NMDA-preferring glutamate receptor biding sites were characterized using the site-selective ligand [3H]MK801, in synaptic membranes prepared from cerebral cortex tissue obtained postmortem from human infants who had died with minimal neurological and neuropathological impairment between 22 and 42 weeks' gestation. It proved necessary to modify the assay protocol used with adult tissue before reliable data could be obtained. In the four cortical region studied (prefrontal, motor, occipital, temporal), [3H]MK801 bound to a single class of sites which showed significant variations in affinity only in motor cortex. The density of [3H]MK801 binding sites (calculated at constant affinity) showed marked increases in all cortical regions over this period. The extent to which glutamate could enhance [3H]MK801 binding became significantly lower in prefrontal and motor cortex as gestation progressed, so that at term, little activation was apparent. In occipital and temporal cortex, this parameter was low throughout late gestation. The evidence suggests that Glutamate-NMDA binding sites may undergo structural rearrangements which alter their ability to interact with ligands during the later stages of human gestation, and that such changes are regionally variable.

The oligomerization by chemical cross-linking of a recombinant human antiviral monoclonal antibody (MAb), r447-1, and its characterization are described. This MAb binds to an epitope residing in the hypervariable V3 region of the envelope protein (gp120/160) of HIV-1. A dimeric form of this MAb displays enhanced avidity and was found to be capable of neutralizing a greater variety of lymphoid cell culture-adapted HIV-1 variants and HIV-1 primary isolates than its monomeric form. The superior binding and breadth of reactivity of this antibody suggests it may have utility as a therapeutic and/or prophylactic agent, if it possesses an appropriate safety and immunogenicity profile.

An analytical method based on radioimmunoassay (RIA) has been developed for the determination of the antiarrhythmic agent, MK-0499, in plasma and urine. Owing to the potency of the drug, the specificity of this assay in human plasma could not be adequately determined using conventional RIA procedures. A highly specific procedure, based on LC/MS-MS, was developed to cross-validate the RIA. The lower quantifiable limits of the RIA and LC/MS-MS-based methods were 0.05 and 0.013 ng ml-1, respectively. Cross-validation data, compared using paired student's t-test regression analysis, showed excellent correlation between methods. The mass spectrometric assay was also used to simultaneously measure plasma concentrations of unlabeled and 14C-labeled MK-0499 following administration of the drug at high specific activity to volunteers.

Peptides representing the N-terminal domain (Ia) of the cardiac sarcoplasmic reticulum protein phospholamban (residues 1-25 [PLB(1-25)] and a phosphorylated form [pPLB(1-25)]) were synthesized and their conformations examined using circular dichroism and nuclear magnetic resonance spectroscopy. In aqueous solution, both PLB(1-25) and pPLB(1-25) adopt a primarily disordered conformation. In 30% trifluoroethanol/10 mM phosphate, PLB(1-25) exhibits a CD spectrum consistent with 60% helical structure. This value decreases to 27% for the phosphorylated peptide. CD spectra in 2% SDS indicate 40% alpha-helix for PLB(1-25) and 20% for pPLB(1-25). Full chemical shift assignments were obtained by conventional homonuclear NMR methodologies for both PLB(1-25) and pPLB(1-25) in 30% trifluoroethanol/water and 300 mM SDS. The solution structure of PLB(1-25) in 30% TFE/water was determined from distance geometry calculations using 54 NOE distance constraints and 17 torsion angle constraints. In the family of 20 calculated conformers, the root mean square deviation from the mean structure is 0.79 A for backbone heavy atoms of residues 1-17. The structure comprises a regular alpha-helix extending from M1 to S16 with the remaining C-terminal residues disordered. The calculated structure is supported by analysis of C alpha H secondary shifts which are significantly negative for residues 1-16. Chemical shift degeneracy is substantially more extensive in the phospho form and precludes a direct comparison of calculated structures. However, the magnitudes of upfield secondary shifts are decreased by 20% in residues 1-11 and are not significantly helical for residues 12-16 according to the criteria of Wishart et al. [(1992) Biochemistry 31, 1647-1651]. 3JHN alpha coupling constants measured for I12, R13, A15, and S16 also suggest that residues 12-16 undergo a local unwinding of the helix upon phosphorylation. Similar results are obtained for PLB(1-25) and pPLB(1-25) in 300 mM perdeuterated sodium dodecyl sulfate except that differences in backbone dynamics for the helical and nonhelical regions of the peptide are evident in the DQF-COSY line shapes for fingerprint cross-peaks. This disruption of structure at the C-terminus of the helix suggests a model for phosphorylation-induced dissociation of the PLB/Ca(2+)-ATPase complex.

The efficacy of nonreamed nailing as the treatment of choice of unstable blunt tibial diaphyseal fractures was studied. From March 1, 1990, through August 31, 1991, 72 patients with 74 fractures that required fixation were treated. One patient died and six were lost to follow-up, leaving 65 patients with 67 fractures. Follow-up averaged 21 months (range 5-43). Fisher's exact and logistic regression analyses were used to compare grades of open fractures, comminution as classified by Winquist, and dynamic and static nailings. The failure rates of 51 titanium and 16 stainless steel nails were compared. Times to union were compared by the log rank statistic method. The average time to union was 32 weeks with 26 (39%) additional operations required to achieve union; 13 dynamizations (12 successful), 12 exchange nailings (11 successful), and one plate and bone graft. The rate of reconstructive procedures to achieve union was a more sensitive indicator of difficulties achieving union than was time to union. Reoperation rates were 33% for closed or grade I and II fractures compared with 46% for grade III fractures (NS). Among closed grade I and II static versus dynamic nailing, times to union were 36 versus 25 weeks (p < 0.01), and the reoperation rates were 44% versus 13% (p < 0.04). Winquist I and II fractures required a 24% reoperation rate versus 53% for grade III and IV and segmental fractures (p < 0.01). Static locked fractures required a 48% reoperation rate versus 12% for dynamic locked fractures (p < 0.01). A logistic regression analysis demonstrated that locking mode was the most important factor in determining reoperation rates. Fifteen additional reoperations for infection, broken or painful implants, or to remodel bones that united with an incomplete circumference of cortex were performed. With an additional 12 elective nail removals, the total reoperations numbered 53 (79%). Titanium alloy nails had a 2% failure rate versus 25% for stainless steel nails (p < 0.01). Two of 28 (7%) grade III fractures became infected. All fractures united within 10 degrees of normal alignment and 1 cm of length. Nine (13%) united with an incomplete cortical circumference, refractory to dynamization and full weight bearing. Thirteen of the 58 (22%) fractures available for an evaluation of ankle motion were symptomatic, with < 10 degrees of dorsiflexion.(ABSTRACT TRUNCATED AT 400 WORDS)

The treatment and results of 13 blunt femoral fractures with an arterial injury were reviewed. Two of the 13 patients (15%) sustained segmental (two levels) arterial injuries. Stabilization of the femur fractures were performed before arterial repair in 10 of the 13 femurs. The results were determined at an average of 4.5 years. For the eight open fractures, two patients had above-knee amputations, no limb regained > 90 degrees of knee motion, four patients required a brace or cane, and three patients have intermittent wound drainage. The five closed fractures all regained full function with full knee motion. Due to the 15% incidence of segmental arterial injury, "wide-field" arteriography is advised for the evaluation of this injury. Femoral stabilization may be performed before arterial repair if the procedure does not encroach on the viability of the limb. The functional results depend on the soft-tissue damage, as illustrated by the poor results seen in open fractures.

Rat liver arginase has been overexpressed in Escherichia coli using a T7-based expression system. The kinetic properties of the recombinant wild-type protein are essentially identical to those of the native rat liver enzyme. The recombinant wild-type protein contains six Mn(II) ions per trimer, in good agreement with results obtained with the fully active native enzyme. However, in contrast to the native enzyme which loses three Mn(II) per trimer upon extended dialysis, the recombinant protein binds Mn(II) tenaciously, and retains six Mn(II) per trimer even after extensive dialysis. Three histidine residues, corresponding to His101, His126, and His141 in the rat liver enzyme, are highly conserved in arginases from evolutionarily divergent species. The replacement of His101 and His126 with Asn by site-directed mutagenesis produced only modest effects on enzymatic activity when measured in the presence of Mn(II) ions. However, EDTA treatment of these mutant enzymes reduced activity to < 0.2% of that for the wild-type enzyme. The activity of wild-type enzyme and the His141 Asn mutant was unaffected by treatment with EDTA. Thus, His101 and His126 are proposed to be ligands to the binuclear Mn(II) center of the enzyme. The His141 Asn mutation produced an enzyme which, in contrast to the native, wild-type, His101 Asn, and His126 Asn arginases, was not inactivated by diethyl pyrocarbonate. These results suggest a catalytic role for His141.

A wide variety of nucleotides are shown to bind to acidic fibroblast growth factor (aFGF) as demonstrated by their ability to (1) inhibit the heat-induced aggregation of the protein, (2) enhance the thermal stability of aFGF as monitored by both intrinsic fluorescence and CD, (3) interact with fluorescent nucleotides and displace a bound polysulfated naphthylurea compound, suramin, (4) reduce the size of heparin-aFGF complexes, and (5) protect a reactive aFGF thiol group. The binding of mononucleotides, diadenosine compounds (ApnA), and inorganic polyphosphates to aFGF is enhanced as the degree of phosphorylation of these anions is increased with the presence of the base reducing the apparent binding affinity. The nature of the base appears to have much less effect. Photoactivatable nucleotides (8N3-ATP, 2N3-ATP, 8N3-GTP, and 8N3-Ap4A) were employed to covalently label the aFGF nucleotide binding site. In general, Kd's in the low micromolar range are observed. Protection against 90% displacement is observed at several hundred micromolar nucleotide concentration. Using 8N3-ATP as a prototypic reagent, photolabeled aFGF was proteolyzed with trypsin and chymotrypsin and labeled peptides were isolated and sequenced resulting in the identification of 10 possible labeled amino acids (Y8, G20, H21, T61, K112, K113, S116, R119, R122, H124). On the basis of the crystal structure of bovine aFGF, eight of the prospective labeled sites appear to be dispersed around the perimeter of the growth factor's presumptive polyanion binding site. On residue (T61) is more distally located but still proximate to several positively charged residues, and another (Y8) is not locatable in crystal structures. Using heparin affinity chromatography, at least three distinct photolabeled aFGF species were resolved. These labeled complexes display diminished affinity for heparin and a reduced ability to stimulate mitogenesis even in the presence of polyanions such as heparin. In conclusion, nucleotides bind apparently nonspecifically to the polyanion binding site of aFGF but nevertheless are capable of modulating the protein's activity. Evidence for the presence of a second or more extended polyanion binding site and the potential biological significance of these results in terms of potential natural ligands of aFGF are also discussed but not resolved.

Peptides and peptide-like molecules as a class have very poor permeability through biological membranes, which severely compromises their potential effectiveness as therapeutic agents. In order to gain insight into the problem of delivering peptide and protein drugs and to establish a model in which the effects of systematic structural variations on transport can be explored, an investigation of the solution conformation of a membrane-permeable peptide was undertaken. Delta-sleep-inducing peptide (DSIP, MW 849) was used in this investigation. DSIP is a charged, hydrophilic peptide that possesses the unusual ability to diffuse passively across the blood-brain barrier (BBB) in vivo [Kastin, A. J., Banks, W. A., Castellanos, P. F., Nissen, C., & Coy, D. H. (1982) Pharmacol. Biochem. Behav. 17, 1187-1191] and across monolayers of brain microvessel endothelial cells in vitro, a model of the BBB [Raeissi, S., & Audus, K. L. (1989) J. Pharm. Pharmacol. 41, 848-852]. This nonapeptide was studied in solution using one- and two-dimensional nuclear magnetic resonance (NMR), circular dichroism (CD), Fourier transform infrared (FT-IR), and fluorescence spectroscopies in conjunction with molecular modeling. Our spectroscopic findings suggest that DSIP exists in a dynamic equilibrium between unordered and folded structures. Residues 2-5 and 6-9 tend to form type I beta-turns in aqueous solution and a similar, but more ordered, helix-like structure inducible in 40% trifluoroethanol (TFE). NMR, FT-IR, and CD studies in aqueous solution support the dynamic equilibrium hypothesis with the IR data, suggesting that the beta-turn population is approximately 40%.(ABSTRACT TRUNCATED AT 250 WORDS)

Forty acute blunt fractures of the humeral diaphysis were treated with Seidel nails (9-mm diameter) between April 1988 and August 1992. Follow up was available for 36 patients. Average Injury Severity Score (ISS) was 22. Forty-eight pelvic and lower extremity fractures necessitated humeral weight bearing in 23 patients. Patients were grouped by canal diameter measured at the point of distal humeral fixation: < or = 9 mm or > or = 10 mm. Data were analyzed with regard to complications, fracture pattern, and time to union. Due to difficulties reaming the humeral canal, five different reaming systems were used. In the < or = 9 mm group (N = 12), there were seven complications (58%). Two patients had iatrogenic comminution distal to the nail. The two open fractures in this group (II, IIIA) developed wound infections, and one, osteomyelitis. Three patients had nonunions: one was lost to follow up at 6 months, and two united at 41 and 74 weeks after exchange nailing and bone grafting. Average time to union was 21 weeks. Six of the seven complications occurred in patients who required reaming of a long, tight segment of distal canal. In the > or = 10 mm group (N = 24), there was one complication of iatrogenic comminution (4%). There were four open fractures (three, grade II; one, IIIB) with no infections. All fractures united (average = 10 weeks). The differences in complications (P = .001) and union (P = .04) between groups were significant. Other complications were not associated with canal diameter or union. There were seven radiographic failures of the distal locking device (19%). Four nails (11%) were left prominent in the shoulder due to technical or equipment failures, and were eventually removed. Four patients (11%) had residual shoulder stiffness (three due to neurologic injury). These primary data suggest use of the Seidel nail is associated with a higher complication rate in humeri with canal diameters < or = 9 mm.

The actions of the anti-ulcer drug sucralfate have been proposed to be mediated through interaction with fibroblast growth factors (Folkman, J., Szabo, S., Strovroff, M., McNeil, P., Li, W. and Shing, Y. (1991) Ann. Surg. 214, 414-427). We show here that acidic fibroblast growth factor (aFGF; FGF-1) binds in vitro to both the soluble potassium salt and the insoluble aluminum salt of sucrose octasulfate, as demonstrated by a variety of biophysical techniques. Similar to the well-described interaction and stabilization of aFGF by heparin, soluble sucrose octasulfate (SOS) stabilizes aFGF against thermal, urea and acidic pH-induced unfolding as determined by a combination of circular dichroism, fluorescence spectroscopy and differential scanning calorimetry. In addition, SOS also enhances the mitogenic activity of aFGF and partially protects the protein's three cysteine residues from copper-catalyzed oxidation. SOS competes with heparin and suramin for the aFGF polyanion binding site as measured by both fluorescence and light scattering based competitive binding assays. Front-face fluorescence measurements show that the native, folded form of aFGF binds to the insoluble aluminum salt of sucrose octasulfate (sucralfate). Moreover, sucralfate stabilizes aFGF against thermal and acidic pH-induced unfolding to the same extent as observed with SOS. Thus, due to their high charge density, SOS and sucralfate bind and stabilize aFGF via interaction with the aFGF polyanion binding site.

At three institutions, 71 humeral intramedullary nails were inserted into the shoulder; 67 were reviewed at 6 months and at completion of treatment. Fifty-one utilized the anterior acromial approach and 16 were inserted lateral to the acromion. Shoulder motion was rated as: excellent (asymptomatic and within 15 degrees of normal); good (normal daily function within normal motion); and poor. Nails were also inserted into the humeral diaphysis of eight cadaver shoulders. Fifty-one nails were inserted via the anterior acromial incision; 48 were graded as excellent, one as good, and two with traumatic axillary neuropathy and reflex sympathetic dystrophy as poor: Sixteen nails were inserted lateral to the acromion; 8 were rated, 7 good, and 1 poor. Motion returned in an average of 17 weeks (range:0-29). The greatest clinical concern is not ultimate shoulder function, but the rate of return. The authors conclude that either the anterior acromial approach or an extraarticular entry portal must be utilized for antegrade humeral diaphyseal nailing.

A combination of near- and far-UV circular dichroism, Fourier-transform infrared spectroscopy, tryptophan fluorescence, size-exclusion chromatography, and a fluorescent extrinsic hydrophobic probe has been employed to characterize partially structured states of human recombinant acidic fibroblast growth factor (aFGF). At low pH, the addition of specific polyanionic ligands or moderate amounts of salts induces states with high secondary but low tertiary structure content. At neutral pH, intermediate amounts of chaotropic agents impose similar partially structured conformational states which also display noncooperative unfolding transitions. Kinetic evidence indicates that similar forms of the protein exist in the first few hundred milliseconds in the refolding pathway of aFGF. The kinetics of their formation appear to be temperature-independent, implying lack of an energy barrier, which is characteristic for further slow folding into the native state. Unlike the native and fully unfolded states, these partially structured conformations exhibit very low solubility, resulting in irreversible aggregation. Potential physiological implications of the existence of such "molten globule" states with regard to the growth factor's transport and biological activity are considered.

The urea-induced unfolding of acidic fibroblast growth factor (aFGF) in the presence and absence of various polyanions has been quantitatively examined by fluorescence spectroscopy. In the absence of a stabilizing polyanion, the apparent free energy of unfolding of aFGF is 6.5 kcal mol-1. The presence of equimolar or greater amounts of heparin stabilizes aFGF from unfolding by more than 2.5 kcal mol-1 and slows the rate of unfolding by greater than 2000-fold. The ability of heparin to stabilize aFGF is critically dependent upon many factors including the number of aFGF molecules bound to the heparin chain, ionic strength, temperature, and the extent of sulfation of the polysaccharide. The presence of similar amounts of other polyanions such as sulfated beta-cyclodextrin or heparan sulfate also stabilizes aFGF to a similar extent as heparin. Additional experiments demonstrate that increasing charge density enhances the ability of polyanions such as sulfated beta-cyclodextrins, phosphorylated inositols, and modified heparins to protect aFGF from urea-induced unfolding.

The peptide (Gly-L-Tyr-L-Asp-L-Lys-L-Tyr)6, referred to as F4-6, was synthesized as a model for a schistosome eggshell protein in which the Gly-Tyr-Asp-Lys-Tyr consensus sequence is repeated over 40 times. Analysis by CD, Fourier transform infrared spectroscopy, potentiometric and spectrophotomertric titrations, NMR, and molecular modeling suggests that F4-6 forms some type of left-handed structure. A likely possibility appears to be a left-handed alpha-helix stabilized by Lysi-Aspi +4 salt bridges and possibly Aspi-Tyri +4 hydrogen bonding and Tyr-Tyr interactions. Spectroscopic studies of a number of F4-6 analogues support this conclusion. For example, substitution of D-Ala for Gly produces a peptide with enhanced left-handed helical spectral characteristics, whereas an L-Ala substitution results in a peptide with minimal structure. These studies suggest that the F4 protein from Schistosoma mansoni may be the first example of a naturally occurring protein devoid of proline and carbohydrate that forms a left-handed helix composed of L-amino acids, although alternative forms of other left-handed structures have yet to be rigorously excluded.

The binding of human acidic fibroblast growth factor (aFGF) to heparin has been analyzed by a variety of different approaches to better elucidate the nature of this protein/sulfated polysaccharide interaction. Static and dynamic light scattering as well as analytical ultracentrifugation analyses indicates that 14-15 molecules of a FGF can bind to a 16-kDa heparin chain, with approximately 10 of these bound relatively uniformly to high-affinity sites. The dissociation constants of these latter sites are estimated to be approximately 50-140 nM on the basis of surface plasmon resonance experiments in which the association and dissociation rates of aFGF interaction with immobilized heparin were measured. The size of the binding site of a FGF on heparin was also determined by heparin lyase digestion of a FGF/heparin complexes followed by isolation and characterization of protected oligosaccharides. The smallest aFGF-protected oligosaccharide comigrated with delta UA2S(1-->4)-alpha-D-GlcNp2S6S(1-->4)-alpha-L-IdoAp-2S( 1-->4)-alpha-D-GlcNp2S6S (where delta UA represents 4-deoxy-alpha-L-threo-hex-4-enopyranosyluronic acid and S is sulfate). Thus, aFGF appears to bind at high density (one molecule every 4-5 polysaccharide units) and with high affinity to heparin. This potentially provides a concentrated, stabilized storage form of the growth factor that can be released for receptor-mediated cellular activation in response to the proper stimuli. It is also possible that close proximity of aFGF molecules on the highly sulfated regions of heparan chains may be involved in the induction of receptor aggregation as suggested by Ornitz et al. [Ornitz, D. M., Yayon, A., Flanagan, J. G., Svahn, C. M., Levi, E., & Leder, P. (1992) Mol. Cell. Biol. 12, 240-247].

The new anticonvulsant vigabatrin (gamma-vinyl-gamma-aminobutyric acid) is normally supplied as a racemate, but its anticonvulsant effect is thought to reside in its [S]-enantiomer only. The plasma concentration ratio of the [R] to [S] enantiomers appears to remain constant across the vigabatrin dosage interval in adult volunteers, and in the present study this has also proved to be the case in 12 chronically treated adult epileptic patients. However, in 8 epileptic children chronically treated with other anticonvulsants and given add-on vigabatrin therapy because of failure to control seizures, plasma [R]:[S]-vigabatrin ratios changed across the drug dosage interval, the [R]-vigabatrin levels tending to be relatively higher soon after intake, and to fall more rapidly than the [S]-vigabatrin concentrations over the next few hours (mean half-lives 2.52 +/- SD 0.49 and 6.53 +/- SD 6.62 hours). The reason for the shorter half-life of [R]-vigabatrin in children remains to be elucidated, but it appears that measurement of racemic vigabatrin plasma concentrations in children, though not in adults, may lead to somewhat misleading conclusions as regards the amount of the circulating anticonvulsant [S]-vigabatrin.

Acidic fibroblast growth factor (aFGF) is markedly stabilized by heparin. Partially due to the heterogeneity of heparin preparations, the nature of the aFGF polyanion binding site is still ill-defined. We have, therefore, investigated a wide variety of well-defined polyanions in terms of their ability to stabilize human recombinant aFGF (15-154) against thermal denaturation. The specificity of the interaction between aFGF and polyanions is shown to be remarkably weak with a surprising number of polyanions (including small phosphorylated and sulfated compounds as well as highly charged biopolymers) able to induce physical stability. Temperature-dependent fluorescence and circular dichroism measurements show that many of these polyanionic compounds stabilize aFGF to the same extent as heparin. The ability of these agents to protect the three free thiol groups of aFGF from copper-catalyzed oxidation was also explored and significant protection was observed. The extent and electrostatic requirements of the protein's polyanion binding site were probed by the use of a series of well-defined heparin fragments and differentially phosphorylated inositol compounds. A tetrasaccharide fragment of heparin is the smallest unit of heparin capable of stabilizing aFGF against thermal denaturation. Increasing phosphorylation of inositol compounds (up to six phosphate groups per molecule) enhances the thermal stability of aFGF. These results are discussed in the context of a model of human aFGF based on the X-ray crystal structure of the bovine protein and previous studies by others of the heparin binding site of both acidic and basic FGF.

The class 2 outer membrane protein (MIEP) of Neisseria meningitidis has recently been shown to be mitogenic for lymphocytes (Liu, M.A., Friedman, A., Tai, J., Martinez, D., Deck, R. R., Hawe, L. A., Shieh, J. T.-C., Jenkins, T. D., Donnelly, J. J., and Oliff, A. I. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 4633-4637. In this study, a possible connection between MIEP's mitogenicity and its possible action as a porin was investigated. MIEP, purified from the bacterial outer membrane protein complex under denaturing conditions, caused a modest but specific release of ions (86Rb) from both erythrocytes and lymphocytes, ultimately resulting in cell lysis. The dose-response of MIEP on erythrocyte lysis was qualitatively similar to a known porin (protein I from Neisseria gonorrhoeae) but was much less efficient. Induction or preservation of native structure in MIEP increased pore formation, resulting in levels comparable to that of the protein I porin. These observations suggest that native MIEP, free of the other outer membrane proteins of Neisseria meningitidis, can efficiently form pores in cells, but that denatured MIEP is variably and marginally effective. However, pore formation by MIEP was not related to its mitogenicity in lymphocytes, based on: (i) native MIEP was not mitogenic; (ii) denatured MIEP was highly mitogenic; and (iii) denatured MIEP was mitogenic at concentrations below the threshold level for pore formation. Therefore, mitogenicity is dependent upon MIEP being in a denatured, monomeric state and is masked by native conformation.

Suramin inhibits the binding of a variety of growth factors to their cell surface receptors. The direct interaction of suramin with acidic fibroblast growth factor has been detected by the enhancement of the drug's fluorescence in the presence of the protein with the maximum effect occurring at a molar ratio of suramin to aFGF of 2:1. This interaction stabilizes aFGF to thermal denaturation and partially protects a free thiol in its polyanion binding site from oxidation. The binding of suramin to aFGF also induces aggregation of the growth factor to at least a hexameric state as detected by static and dynamic light scattering as well as by gel filtration studies. Both CD and amide I' FTIR spectra of aFGF in the presence and absence of suramin suggest that the drug may also be causing a small conformational change in the growth factor. Suramin produces an even greater aggregation of bFGF and PDGF but not of EGF or IGF-1. Evidence for a suramin-induced conformational change in IGF-1 but not EGF is found by CD, however. It is concluded that suramin binds to many growth factors and that this induces microaggregation and, in some cases, conformational changes. In the case of aFGF, suramin interacts at or near its heparin binding site. The relationship between these phenomena and the anti-growth factor activity of suramin remains to be clearly elucidated.

From January 1982 through December 1988, 150 patients with 153 Winquist Class III and IV comminuted diaphyseal femur fractures due to high energy blunt trauma were treated with immediate plate fixation. A total of 260 major general surgical systems were injured in 150 patients. Forty-nine patients did not have adequate preoperative spine radiographs due to positioning or time factors. Nineteen patients had spine fractures; nine were diagnosed post-femoral fixation. The average injury severity score (ISS) was 22.7. Three patients died (2%). Our institution predicted mortality with this ISS for patients without pelvic or femur fractures at 15% (P = .0003). Six patients moved to other states and three were lost to follow up due to noncompliance. One of us reviewed 141 fractures in 138 patients at a minimum of 12 months follow up and completion of treatment. Forty-nine fractures were open; 8 grade I, 25 grade II, 10 grade IIIA, 4 IIIB, 2 IIIC. A total of 153 pelvic or ipsilateral major orthopedic injuries were present in 141 fractures. An additional 188 major associated orthopedic injuries were noted. The average time to union was 17.2 weeks. One plate was applied in 11 degrees of varus. Five plates failed from fatigue and five from repeat traumas. Seven plate failures were rodded and healed within 8 weeks. There was one persistent nonunion. One fracture, open IIIC, became infected after uniting. One patient has 110 degrees of knee motion and 140 fractures have greater than 130 degrees of knee motion. Plate fixation is a safe technique for immediate femoral stabilization in the face of high energy blunt trauma. Failures occur late and are easy to reconstruct. Intramedullary nails are the preferred method of reconstruction. Ultimate knee function is excellent. Infection rates (1/49) in open fractures are acceptably low.

The N-terminal domain of human immunodeficiency virus (HIV-1) integrase (IN) contains the sequence motif His-Xaa3-His-Xaa23-Cys-Xaa2-Cys, which is strongly conserved in all retroviral and retrotransposon IN proteins. This structural motif constitutes a putative zinc finger in which a metal ion may be coordinately bound by the His and Cys residues. A recombinant peptide, IN(1-55), composed of the N-terminal 55 amino acids of HIV-1 IN was expressed in Escherichia coli and purified. Utilizing a combination of techniques including UV-visible absorption, circular dichroism, Fourier transform infrared, and fluorescence spectroscopies, we have demonstrated that metal ions (Zn2+, Co2+, and Cd2+) are bound with equimolar stoichiometry by IN(1-55). The liganded peptide assumes a highly ordered structure with increased alpha-helical content and exhibits remarkable thermal stability. UV-visible difference spectra of the peptide-Co2+ complexes directly implicate thiols in metal coordination, and Co2+ d-d transitions in the visible range indicate that Co2+ is tetrahedrally coordinated. Mutant peptides containing conservative substitutions of one of the conserved His or either of the Cys residues displayed no significant Zn(2+)-induced conformational changes as monitored by CD and fluorescence spectra. We conclude that the N terminus of HIV-1 IN contains a metal-binding domain whose structure is stabilized by tetrahedral coordination of metal by histidines 12 and 16 and cysteines 40 and 43. A preliminary structural model for this zinc finger is presented.

Recombinant tick anticoagulant peptide (r-TAP), a potent and specific inhibitor of blood coagulation factor Xa, was purified to greater than 99% homogeneity at the multi-gram scale. Genetically engineered yeast secreted 200-250 mg/l of the heterologous protein into the medium. Cells were separated from broth by diafiltration and purification was done by two chromatographic steps, both conducive to operation on a large scale. Analysis of the purified protein by several methods indicated that it was greater than 99% homogeneous and no incompletely processed or truncated proteins were detected. Physico-chemical characterization data of r-TAP show that it exists as a monomer in solution and no evidence of post-translational modification was observed. The purified protein was fully active in inhibiting human coagulation factor Xa.

Described here is a young woman with intermittent Cushing's disease associated with psychotic disturbance. On five occasions, over a period of four years, she became severely depressed and had acne, amenorrhea, hirsuties and moon facies. On each of the first four occasions the symptoms lasted from two to three months and disappeared spontaneously. Biochemical investigations, including plasma ACTH estimations during the last two episodes, indicated that the adrenocortical overactivity was pituitary-dependent. During the fifth relapse the patient experienced a very severe, depressive psychosis, necessitating bilateral adrenalectomy, which resulted in complete remission.