Protein fixation and antigen retrieval: chemical studies.

Abstract Fixation with formaldehyde is the first process to which most biopsy and necropsy specimens are exposed prior to dehydration and embedding in paraffin wax. Tissue specimens that have been fixed in formaldehyde have architectural characteristics that are familiar to virtually every pathologist and these facilitate routine diagnosis. Nevertheless, formaldehyde fixation has some deleterious effects including reduction in immunoreactivity and degradation of nucleic acids. Development of methods to counteract these deleterious effects requires an understanding of the chemical events that occur during tissue fixation and subsequent tissue processing. This short review illustrates some of the chemical consequences of formaldehyde fixation and ethanol dehydration. It also provides some insight into the molecular events accompanying heat-induced antigen retrieval.

Redox properties of the Rhodobacter sphaeroides transcriptional regulatory proteins PpsR and AppA.

Redox properties of the photosynthetic gene repressor PpsR and the blue-light photoreceptor/antirepressor AppA from Rhodobacter sphaeroides have been characterized. Redox titrations of PpsR reveal the presence of a two-electron couple, with an E (m) value of -320 mV at pH 7.0, which is likely to arise from the reversible conversion of two cysteine thiols to a disulfide. This E (m) value is very much more negative than the E (m) = -180 mV value measured previously at pH 7.0 for the disulfide/dithiol couple in CrtJ, the homolog for PpsR in the closely related bacterium Rhodobacter capsulatus. AppA, a flavin-containing blue-light receptor that is also involved in the regulation of gene expression in R. sphaeroides, contains multiple cysteines in its C-terminal region, two of which function as a redox-active dithiol/disulfide couple with an E (m) value of -325 mV at pH 7.0 in the dark. Titrations of this dithiol/disulfide couple in illuminated samples of AppA indicate that the E (m) value of this disulfide/dithiol couple is -315 mV at pH 7.0, identical to the value obtained for AppA in the dark within the combined experimental uncertainties of the two measurements. The E (m) values of AppA and PpsR demonstrate that these proteins are thermodynamically capable of electron transfer for their activity as an anti-repressor/repressor in R. sphaeroides.

Interleukin-2-induced small unilamellar vesicle coalescence.

Recombinant human interleukin-2 (rhIL-2) was incorporated in liposomes for potential therapeutic applications using a novel process. In this process, rhIL-2 caused the formation of large, unique multilamellar vesicles (MLVs) from small unilamellar vesicles (SUVs) of dimyristoylphosphatidylcholine (DMPC). Vesicle coalescence occurred most rapidly at 19 degrees C, between the pre- and main phase transition temperatures of DMPC, and showed a dependence upon pH (pH <5.5), ionic strength (>50 mM) and the initial size of the unilamellar vesicles (

Investigation of phase transitions in bilayer membranes.

This article described three techniques used to study phase transitions in phospholipid bilayers. The complementarity of the three techniques in characterizing the thermotropic and structural properties of phospholipid bilayers has been demonstrated by describing their use to characterize a series of mixed-chain-length PCs. It has been shown that an understanding of the energetics that govern the packing of phospholipid chains in the gel phase can be used to construct a model to interpret thermodynamic data of the PCs. This model, in turn, provided a framework for designing and interpreting the Raman spectroscopic and X-ray diffraction experiments on this series of phospholipids. The result was a complete description of the phase transitions and gel phase packing properties of the mixed-chain-length PCs. The phase diagram of Fig. 5B has been expanded to include the mixed-chain-length PC series C18C18PC through C18C0PC. Furthermore, the phase diagram and the chain inequivalence parameter have been shown to describe the behavior of any mixed-chain-length PC, irrespective of the lengths of the hydrocarbon chains or the position of the chains on the glycerol backbone. This is demonstrated by the additional mixed-chain-length PCs plotted in Fig. 5B. With minor modifications, the phase diagram also accurately describes the behavior of mixed-chain-length phosphatidylethanolamines, sphingomyelins, and unsaturated PCs. Finally, it has been demonstrated that a correlation exists between the thermodynamic and the Raman spectroscopic parameters determined for the phase transition of phospholipid bilayers. This correlation is based on the common chain energetics being measured by these two techniques.

Packing characteristics of two-component bilayers composed of ester- and ether-linked phospholipids.

The miscibility properties of ether- and ester-linked phospholipids in two-component, fully hydrated bilayers have been studied by differential scanning calorimetry (DSC) and Raman spectroscopy. Mixtures of 1,2-di-O-hexadecyl-rac-glycero-3-phosphocholine (DHPC) with 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DHPE) and of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) with 1,2-di-O-hexadecyl-sn-glycero-3-phosphoethanolamine (DHPE) have been investigated. The phase diagram for the DPPC/DHPE mixtures indicates that these two phospholipids are miscible in all proportions in the nonrippled bilayer gel phase. In contrast, the DHPC/DPPE mixtures display two regions of gel phase immiscibility between 10 and 30 mol% DPPE. Raman spectroscopic measurements of DHPC/DPPE mixtures in the C-H stretching mode region suggest that this immiscibility arises from the formation of DHPC-rich interdigitated gel phase domains with strong lateral chain packing interactions at temperatures below 27 degrees C. However, in the absence of interdigitation, our findings, and those of others, lead to the conclusion that the miscibility properties of mixtures of ether- and ester-linked phospholipids are determined by the nature of the phospholipid headgroups and are independent of the character of the hydrocarbon chain linkages. Thus it seems unlikely that the ether linkage has any significant effect on the miscibility properties of phospholipids in biological membranes.

Lamellar-phase polymorphism in interdigitated bilayer assemblies.

Bilayers composed of 1-octadecanoyl-2-decanoyl-sn-glycero-3-phosphocholine (C(18)C(10)PC) adopt a mixed-interdigitated gel-phase packing where the short chains of the C(18)C(10)PC molecules pack end-to-end while their long chains span the entire hydrocarbon width of the bilayer. Calorimetric cooling scans of freshly prepared hand-shaken bilayer suspensions of C(18)C(10)PC exhibit a single exothermic phase transition at 14.6 degrees C, whereas suspensions incubated at temperatures below 2 degrees C for several days exhibit an additional phase-transition exotherm at 17.9 degrees C. Calorimetric and electron microscopic evidence is presented that low-temperature incubation of C(18)C(10)PC bilayer suspensions composed of liposomes of heterogeneous size leads to the conversion of those liposomes in the suspension below about 0.2 microns in diameter into planar lamellar sheets. These lamellar sheets are the origin of the phase-transition exotherm at 17.9 degrees C, whereas the phase-transition exotherm at 14.6 degrees C arises from the liposomes in the suspension. We also show that phosphatidylcholine bilayer suspensions, induced to interdigitate by ethanol, exhibit a similar thermotropic behavior. The implication of these findings for the reversibility of interdigitated gel to liquid-crystalline phase transitions and the role of phospholipid molecular geometry in the formation of interdigitated bilayers are addressed.

Properties of phosphatidylcholine bilayers as revealed by mixed-acyl phospholipid fluorescent probes containing n-(9-anthroyloxy) fatty acids.

A series of five mixed-acyl phosphatidylcholine (PC) fluorescent probes having the structure 1-caproyl-2-(n-(9-anthroyloxy)-acyl)-sn-glycero-3-phosphocholine, where the sn-2 anthroyloxy-labeled acyl chain is stearic acid (n = 2,6,9,12) or palmitic acid (n = 16), have been prepared. These probes have been used to study the thermal behavior and transbilayer organization of 1,2-distearoyl-PC (C(18)C(18)PC), 1-stearoyl-2-caproyl-PC (C(18)C(10)PC), and 1-caproyl-2-stearoyl-PC (C(10)C(18)PC) multilamellar dispersons. These probes reported the noninterdigitated gel to liquid-crystalline phase transition of C(18)C(18)PC at 55.1 degrees C and the mixed-interdigitated gel to liquid-crystalline phase transitions of C(18)C(10)PC and C(10)C(18)PC at 19.1 and 10.1 degrees C, respectively. The results suggest that, upon cooling, the C(18)C(10)PC liquid-crystalline phase transforms to the mixed-interdigitated gel phase by way of a partially interdigitated gel-phase intermediate. Isothermal plots of anisotropy versus the position of the anthroyl moiety on the sn-2 acyl chain of the PC probes were used to construct transbilayer anisotropy profiles of the gel phases of the three bilayer systems. These anisotropy profiles can serve as 'interdigitation signatures' that clearly distinguish the noninterdigitated from the mixed-interdigitated gel-phase bilayer organization. In the liquid-crystalline phase, the anisotropy profiles suggest that the dynamic motions of the disordered acyl chains of the mixed-acyl PCs are influenced by the interpenetration of the chains from the opposing leaflets of the bilayer.

Effects of formaldehyde fixation on protein secondary structure: a calorimetric and infrared spectroscopic investigation.

We investigated the effects of formaldehyde fixation on the secondary structure of isolated proteins (bovine serum albumin, ribonuclease A, and hemoglobin) using high-sensitivity differential scanning calorimetry and Fourier transform infrared spectroscopy. Whereas thermograms obtained by scanning calorimetry on unfixed purified proteins demonstrated denaturation transitions in the 70-90 degrees C temperature range, the thermograms showed no denaturation transitions in this temperature range when the proteins had been placed in formaldehyde solutions. Thus, fixation destroyed the denaturation transition of bovine serum albumin, ribonuclease A, and hemoglobin. Infrared spectra obtained on the unfixed and fixed proteins were essentially identical. This demonstrates that the "fixed" proteins retain the secondary structure present before fixation. We therefore conclude that the cross-linking of proteins that occurs in the process of formaldehyde fixation "locks in" the secondary structure of these protein molecules.

Simultaneous determination of sulfasalazine and its metabolites sulfapyridine and N-acetylsulfapyridine in human serum by ion-pair high-performance liquid chromatography using a polymer-based column.

An HPLC method is described for the simultaneous and rapid determination of sulfasalazine (salicylazosulfapyridine) and two of its metabolites, sulfapyridine and N-acetylsulfapyridine, in human serum. The range of quantitation is 0.1 to 12 micrograms/mL for sulfasalazine and sulfapyridine and 0.25 to 12 micrograms/mL for N-acetylsulfapyridine. Serum is mixed with acetonitrile containing the internal standard sulfamethazine and the ion-pairing agent tetraethylammonium chloride. The acetonitrile extract is concentrated and analyzed by HPLC, using a new polymer-based column, and detected by UV spectroscopy at 270 nm. This paper is the first both to describe the simultaneous analysis of all three of the compounds from serum and to present sulfasalazine concentration-time data following oral administration to humans.

Adult myelography with iohexol.

An open, multicenter trial of adult myelography was carried out at six centers in 117 patients with iohexol (Omnipaque) 240 mg I/ml and 300 mg I/ml to determine whether there was a difference in visualization or side-effects when a lumbar or cervical approach was used to visualize the areas where disease was suspected. One hundred and thirteen myelograms were analysable. In over 85% of the myelograms with a lumbar approach, visualization of the area injected was excellent or good. Just over half of these were obtained with the intention of visualizing primarily the cervical area, and in 93% of these visualization was good or excellent at that site. In 100% of the cervically injected myelograms, visualization was excellent in the cervical area. Areas more distant from the injections were well visualized in the majority of patients. There was no significant difference overall in visualization with either of the two concentrations of iohexol used. There were 59 side-effects, mostly mild in nature; 40 of them occurred in 50 patients receiving the higher concentration and 19 in 63 patients receiving the lower concentration. The most common side-effect was headache (23 patients). Nystagmus was the only severe side-effect and occurred in only one patient having cervical myelography. The patient made a complete recovery within 36 hours.

Effects of headgroup methylation and acyl chain length on the volume of melting of phosphatidylethanolamines.

The change in volume associated with the gel to liquid-crystalline phase transition for phosphatidylethanolamines of various chain lengths and headgroup methylation was determined by measuring the pressure dependence of the phase transition temperature and computing the volume change by using the Clausius-Clapyron equation. The volumes thus obtained were comparable to those previously obtained by using scanning dilatometry. The melting volume was larger for lipids with longer acyl chains, as found previously. The melting volume for a series of N-methylated dipalmitoylphosphatidylethanolamines (DPPEs) did not increase monotonically with increasing headgroup methylation. Instead, the melting volume increased in the order N,N-dimethyl-DPPE less than N-methyl-DPPE less than DPPE less than dipalmitoylphosphatidylcholine. This unanticipated result is hypothesized to result from the competing effects of headgroup methylation on molecular volume and hydrogen bonding on the volume of melting.

Thermotropic properties of saturated mixed acyl phosphatidylethanolamines.

The mixed acyl phosphatidylethanolamine (PE) series C(18)C(18)PE, C(18)C(16)PE, C(18)C(14)PE, C(18)C(12)PE, and C(18)C(10)PE has been prepared from the corresponding phosphatidylcholines by phospholipase D mediated transphosphatidylation. The thermotropic behavior of unhydrated and hydrated preparations of these PEs has been investigated by differential scanning calorimetry and 31P NMR spectroscopy. Unhydrated preparations of the PEs undergo crystalline to liquid-crystalline transitions (Tm+h), which correspond to the simultaneous hydration and acyl chain melting of poorly hydrated crystalline samples. Hydrated preparations of the PEs undergo gel to liquid-crystalline transitions (Tm) when scanned immediately subsequent to cooling from temperatures above their respective Tm+hs. Multilamellar bilayers of C(18)C(18)PE, C(18)C(16)PE, and C(18)C(14)PE pack without significant interdigitation of the phospholipid acyl chains across the bilayer center in the gel phase. C(18)C(10)PE multilamellar preparations exhibit a mixed-interdigitated gel phase packing of the phospholipid acyl chains. Hydrated bilayers of C(18)C(12)PE adopt a mixed-interdigitated gel phase packing at temperatures below 13.9 degrees C. Between 13.9 degrees C and the gel to liquid-crystalline transition temperature of 36.9 degrees C, the C(18)C(12)PE bilayer adopts a noninterdigitated gel phase packing. The metastable behavior of fully hydrated and partially hydrated preparations of the mixed acyl PEs has been investigated. Bilayers of C(18)C(18)PE, C(18)C(16)PE, and C(18)C(14)PE exhibited little or no tendency toward regeneration of the crystalline phase. In contrast, bilayers of C(18C(12)PE and C(18)C(10)PE exhibited a metastability of the liquid-crystalline phase in the temperature interval between Tm and Tm+h, which can allow for the regeneration of the crystalline phase under certain conditions. Bilayers of C(18)C(12)PE exhibited an additional metastability of the noninterdigitated gel phase.

Risk factors for urethral injuries in men with traumatic pelvic fractures.

Urethral injuries are commonly associated with pelvic fractures. The prompt recognition and appropriate management of these injuries may significantly impact subsequent morbidity, yet few studies have addressed the identification of the risk factors for urethral injury in men with pelvic fractures. We reviewed retrospectively the records of 405 men with pelvic fractures seen at our medical center, including 21 (5 per cent) with urethral injuries. Of the 21 men 14 (67 per cent) had fractures involving a pubic ramus and a sacroiliac joint, and 12 (57 per cent) had no physical signs (blood at the urethral meatus, perineal hematoma or a high-riding prostate) that would suggest a urethral injury. The likelihood for the presence of physical signs is directly related to the interval since injury. We believe that men with the combination of rami fractures and sacroiliac disruption should undergo retrograde urethrograms before urethral instrumentation, and that physical signs are unreliable indications for urethral injuries, especially soon after the injury.

Mixing behavior of symmetric chain length and mixed chain length phosphatidylcholines in two-component multilamellar bilayers: evidence for gel and liquid-crystalline phase immiscibility.

The mixing behavior of symmetric chain length and mixed chain length phosphatidylcholines in two-component multilamellar bilayers has been investigated by high-sensitivity differential scanning calorimetry. Phase diagrams have been constructed for two-component bilayers composed of C(18)C(18)PC and either C(18)C(16)PC, C(18)C(14)PC, C(18)C(12)PC, or C(18)C(10)PC. It is found that C(18)C(18)PC-C(18)C(16)PC and C(18)C(18)PC-C(18)C(14)PC mixed bilayers exhibit complete miscibility of the components in both the gel and liquid-crystalline phases. Whereas this mixing is observed to be nearly ideal for the C(18)C(18)PC-C(18)C(16)PC binary system, the intermixing of the lipids is highly nonideal in the gel phase of the C(18)C(18)PC-C(18)C(14)PC binary mixture. The C(18)C(18)PC-C(18)C(12)PC and C(18)C(18)PC-C(18)C(10)PC mixed bilayers are characterized by partial immiscibility of the phosphatidylcholine components in the bilayer gel phase. Over a large compositional range, these bilayers appear to consist of phase-separated regions of interdigitated and noninterdigitated gel phases. In addition, the C(18)C(18)PC-C(18)C(10)PC two-component bilayer displays a limited region of liquid-liquid immiscibility in the liquid-crystalline bilayer phase. The phase separation of the mixed chain length phosphatidylcholines revealed in these mixed bilayers may represent a three-dimensional phase separation of the lipid components where the phosphatidylcholines are both laterally separated within the plane of the bilayer and conformationally coupled across the bilayer. Such phase-separated domains could have profound effects on membrane structure and function if they were to occur in biological membranes.

Calorimetric and fatty acid spin label study of subgel and interdigitated gel phases formed by asymmetric phosphatidylcholines.

Several saturated asymmetric and symmetric phosphatidylcholines were studied by ESR spectroscopy and differential scanning calorimetry in order to determine the behavior of a fatty acid spin labeled near its terminal methyl, 16-doxylstearate, in the mixed interdigitated gel phase and the Lc subgel phase and other properties of these lipids. This spin label was motionally restricted in the mixed interdigitated gel phases of 18:10PC and 18:12PC. The motional restriction was similar to that reported earlier for fully interdigitated phases. This spin label was motionally restricted almost to the same degree in 10:18PC suggesting that this asymmetric lipid may also form a mixed interdigitated bilayer. In contrast the spin label had more motion in the gel phase of 18:14PC than in symmetric forms of PC, consistent with conclusions from X-ray diffraction studies that this less asymmetric lipid does not form a mixed interdigitated phase. The spin label was partially frozen out of the Lc subgel phases of symmetric forms of PC and 18:14PC formed by storage at low temperature. The phase behavior of the other asymmetric lipids also depended on the sample history. Storage at low temperature caused 10:18PC and 18:12PC to go into ordered phases. The enthalpy of the transition of these ordered phases to the liquid-crystalline phase was 2-2.4-times greater than that of the transition of the gel phase formed on cooling back from the liquid-crystalline phase. The temperature of this high enthalpy transition was 0.8 K below that of the lower enthalpy gel to liquid-crystalline phase transition for 18:12PC, but 4.6 K higher for 10:18PC. The spin label was frozen out of these ordered phases, as it was out of the Lc subgel phases, suggesting that 18:12PC and 10:18PC may also form an Lc phase. 18:10PC was not observed to form an ordered phase although storage of the sample at low temperatures did affect the temperature of its transition from the liquid-crystalline phase back to the gel phase upon cycling through its phase transition.

Urinary calculous disease in Southeast Asian immigrants.

Recent immigrants from Southeast Asia accounted for 39 of 149 hospital admissions for treatment of urinary tract stones. Presumptive diagnosis of a urinary calculus was possible in only 19 per cent of the refugees compared with 60 per cent of other patients treated at the same hospital (p less than 0.005). Calculi in Southeast Asian immigrants were larger (p less than 0.001), and surgical procedures were required more often (p less than 0.05) than for other patients with calculi. Urinary stones should be considered a likely cause of abdominal or urinary tract complaints in recent immigrants from Southeast Asia.

Polymorphic phase behavior of platelet-activating factor.

Vibrational Raman and 31P NMR spectroscopic experiments have been performed as a function of temperature on aqueous dispersions of 1-0-octadecyl-2-acetoyl-sn-glycero-3-phosphocholine, a chemically synthesized platelet-activating factor. In the temperature range of -7 to 30 degrees C, the C(18)/PAF-H2O system is shown, upon heating, to undergo two thermal phase transitions centered at 9.2 degrees and 18.4 degrees C. The low temperature transition, attributed to the interdigitated lamellar gel (II)----gel (I) phase transition, is characterized by the breakdown of large lamellar organizations into small, but aggregated, bilayer vesicles. The high-temperature transition corresponds to the interdigitated lamellar gel (I)----micellar transition. The molecular ordering and packing structure of C(18)/PAF in the two lamellar phases and phase transition regions are described. It appears that the interdigitated lamellar gel (I) phase is unique for C(18)/PAF dispersions when compared with the behavior of other chemically closely related phospholipids in excess water.

Adjuvant radiotherapy following radical prostatectomy: results and complications.

Between 1954 and 1978, 148 patients underwent radical perineal prostatectomy for adenocarcinoma clinically confined to the prostate gland. This report is based on 45 of these patients with microscopic extension of disease beyond the gland and a minimum 5-year followup. Of the patients 22 received adjuvant external beam radiation therapy and 23 did not. The groups were comparable with regard to significant prognostic variables. Patient selection was by surgeon preference. Local recurrences were seen in 1 of 22 patients (5 per cent) receiving adjuvant radiotherapy and 7 of 23 (30 per cent) undergoing an operation alone (p less than 0.05). Of 8 patients with local recurrence 7 died of the disease. Delayed radiotherapy of a local recurrence generally was not effective in controlling the disease. Of the 11 patients who died of prostatic cancer with a mean followup of 9.2 years 3 received adjuvant radiotherapy and 8 did not. Severe but nonfatal long-term complications were seen in 14 per cent of the irradiated patients and 6 per cent of those treated with an operation alone. Most of the complications occurred in the earlier years of the study in patients who received 60cobalt radiotherapy. When clinical stage B cancer of the prostate is found to be pathological stage C following radical perineal prostatectomy, adjuvant radiotherapy can decrease the incidence of subsequent local recurrence. The potential risk of adjuvant radiation therapy should be weighed and its use considered, particularly in patients whose tumor extends to the surgical margins or who have seminal vesicle invasion.