Bovine hemoglobin as a potential source of hemoglobin-based oxygen carriers: crosslinking with bis(2,3-dibromosalycyl)fumarate.

Reaction in anaerobic conditions of bovine hemoglobin with bis(2,3-dibromosalycyl)fumarate resulted in new derivatives with P50 in excess of 40 mmHg, as determined at 37 degrees C in 0.15 M Cl- at pH 7.4. Although the chromatographic preparations indicated some heterogeneity of the reacted material, the proteins obtained were homogeneous with regard to sedimentation velocity, which showed the presence of only nondissociable tetrameric species. SDS gel electrophoresis showed the presence of a new band with a mobility corresponding approximately to a molecular mass of 32 kDa, indicating the presence of covalent intramolecular crosslinks between subunit pairs. Chromatographic analyses indicated that both alpha and beta chains were chemically modified. The retention times in rats of the crosslinked hemoglobin was 10-times longer than that of untreated hemoglobin.

Renal excretion of pseudo-cross-linked human, porcine and bovine hemoglobins.

UNLABELLED: The present experiments were done to determine plasma retention, urinary excretion, and acute renal effects of bolus administration (20 mg/100 g), of hemoglobins (human, bovine and porcine) whose tetrameric structure was stabilized by a pseudo-cross-link between the beta-chains. Standard renal clearance studies showed that these preparations had no immediate adverse effects on GFR or ERPF, however, urine flow and electrolyte excretion commonly increased. Plasma disappearance curves best fit a double-exponential and showed components with half-times of 28 +/- 3 and 257 +/- 43 min. A variable fraction, 57 +/- 6%, appeared in the urine, in phase with the early-fast component of plasma disappearance. This in vivo finding of extensive urinary excretion conflicted with sedimentation velocity analysis of these preparations which indicated a homogenous preparation with mean molecular weight of a stable and therefore, unfilterable tetramer. It appears that the glomerular filter selects for permeant species not easily detected by sedimentation velocity analysis. The different species may reflect the presence, within the PXLs of conformational-slow-equilibria among isomeric forms with different degrees of filterability. IN CONCLUSION: It is important, useful and simple to determine urinary excretion as a means to ascertain molecular stability in vivo.

Modulation of oxygen affinity in hemoglobin by solvent components. Interaction of bovine hemoglobin with 2,3-diphosphoglycerate and monatomic anions.

In the absence of Cl- in Hepes buffer at pH 7.4, the oxygen affinity of bovine and human hemoglobin is equally sensitive to 2,3-diphosphoglyceric acid. The low oxygen affinity measured for bovine hemoglobin at physiological salt concentration can be explained by the high affinity of Cl- anions for oxygen-linked sites that are absent in human hemoglobin. Bovine hemoglobin can discriminate between the different halogens in the sense that different halide concentrations are necessary to produce the same P50. Competition experiments indicate that the halogens interact with the same oxygen-linked sites. In agreement with the different affinities for halides, the Bohr effect of bovine hemoglobin is larger in the presence of Cl- than in that of Br- and there is good agreement between the number of protons and anions exchanged with the solvent upon oxygenation of bovine hemoglobin.

Primary adenocarcinoma of the angle of Treitz. Case report and review of the literature.

We describe a 54-year-old man who was referred with the diagnosis of adenocarcinoma of the angle of Treitz and treated with the use of a duodenojejunal segmentary resection. After a review of the literature, we emphasize the difficulty of making an early diagnosis and discuss the most appropriate surgical treatment of tumors of the angle of Treitz. The better prognosis of these neoplasms compared with those of the proximal duodenum can be supported by some embryological findings observed from a different research project currently in progress.

A possible new mechanism of oxygen affinity modulation in mammalian hemoglobins.

Bovine red cells do not contain appreciable amounts of 2,3-diphosphoglycerate (2,3-DPG). Bovine hemoglobin, however, has a particular sensitivity to chloride ions and as a result it can attain oxygen affinity values lower than those measured for human hemoglobin in the presence of 2,3-DPG. The interaction of bovine hemoglobin with anions is modulated by the hydrophobic characteristics of the protein. Comparison of the hydropathy plots of primate and ruminant hemoglobins indicates constant regions of opposite hydrophobicity, which have fixed amino acid differences. A model is proposed for explaining the regulation of oxygen affinity by chlorides, as an alternative to the classic modulation by 2,3-DPG.

Circular dichroism kinetics of acid denaturation of hemoglobin and of its beta-subunits.

Human hemoglobin and its isolated beta-subunits were denatured by addition of HCl so as to reach final pH values ranging from 2.0 to 3.2. The beta-subunits were alkylated in both the beta 93 and beta 112 cysteines; this treatment makes the beta-subunits monomeric. The kinetics of acid denaturation of the two proteins was followed spectropolarimetrically in the millisecond time range, measuring the changes in circular dichroism at 225 nm. At all pH values, in both systems, the decay of ellipticity could be simulated by two exponentials. The initial ellipticity values of the solutions, obtained by extrapolation at zero time, were those expected for the native proteins. The rates of denaturation were lower in the hemoglobin system than in the isolated monomeric beta-subunits. The data suggest that in the tertiary structure of hemoglobin and beta IAA there are different domains which unfold at different rates upon exposure to acid.

Local phenomena and distribution of molecular species during the unfolding of heme-free myoglobin in the presence of GdnHCl and urea as seen by time-resolved fluorescence spectroscopy.

We have used time-resolved fluorescence spectroscopy for following the unfolding of apomyoglobin in urea and guanidine hydrochloride (GdnHCl). The data have been compared with those obtained using classical techniques such as CD and steady-state emission spectroscopy. Both the average intensity of the lifetimes and the size of the librational cone of the fluorophores, as measured by time-resolved fluorescence, increased with denaturant concentration and their changes largely preceded the modifications detectable with CD and the shift of the maximum of emission spectra. The data indicate that the changes in the local environments of the tryptophans were completed when the global modification monitored by CD and the emission spectra was still minimal. This suggests that an initial event in the denaturation of apomyoglobin is localized at the tryptophan residues. The correlation times of native apomyoglobin showed the rotational diffusion characteristics of a rigid rotor. In 3.6 M GdnHCl and 7.5 M urea, where the secondary structure is practically absent, the correlation times of the two systems became very short, as expected from the motion of a flexible polymer. In GdnHCl, under conditions of partial unfolding, it was not possible to detect the presence of native totally folded molecular species.

Solvent perturbation evidence for a two-state system regulated by calcium in sarcoplasmic reticulum ATPase.

Perturbation of sarcoplasmic reticulum ATPase with the nonionic detergent C12E8 is modulated by the amount of free Ca2+ present in the solvent prior to the addition of detergent. CD measurements show that the enzyme exists in solution in two different conformations that react differently with the detergent. They probably represent the free enzyme, and its complex with Ca2+. On this assumption, titrations with increasing amounts of Ca2+ produced data superimposable on curves obtained measuring Ca2+ bound to sarcoplasmic reticulum vesicles.

Librational motions of membrane-embedded Ca2+-ATPase of sarcoplasmic reticulum labeled with fluorescein isothiocyanate.

Continuing our investigation of the relationships between internal motions and functional properties of soluble and membrane-bound proteins we have explored the lifetimes and correlation times associated with the fluorescence emission of fluorescein-labeled Ca2+-dependent ATPase of sarcoplasmic reticulum. The emission was characterized by two lifetime components near 1.8 and 4.1 ns, probably due to exposure of the probe to environments of different polarities. The time-dependent anisotropy showed the presence of two correlation times near 0.8 and 6.6 ns. The shorter correlation time was due to motions of the probe around its point of attachment on the surface of the protein. The longer correlation time indicated the presence of internal motions of the protein. Both lifetimes and correlation times were insensitive to temperature between 2 and 10 degrees C. They were also insensitive to addition and removal of 100 microM free Ca2+.

Effect of temperature on oxygen affinity and anion binding of bovine hemoglobin.

Measurements of oxygen binding to bovine hemoglobin have been carried out over the temperature range 15-37 degrees C at pH 7.33. The standard enthalpy of oxygenation after correction for the heat of oxygen solution and of the Bohr protons is found to be -7.1 or -7.2 kcal/mol in the presence of 0.1 M chloride or bromide, respectively. This value is well below the -14.4 kcal/mol determined for human hemoglobin under identical experimental conditions. As reported by Fronticelli et al. (C. Fronticelli, E. Bucci and A. Razynska, J. Mol. Biol. 202 (1988) 343), the preferential binding of anions by bovine hemoglobin recognizes the various halides. Measurements at various temperatures reveal that this is true only above 25 degrees C. The halide recognition and the less exothermic enthalpy of oxygenation of bovine hemoglobin are probable due to oxygen-linked hydrophobic effects that are larger in bovine than in human hemoglobin.

The dimer-tetramer equilibrium of recombinant hemoglobins. Stabilization of the alpha 1 beta 2 interface by the mutation beta(Cys112-->Gly) at the alpha 1 beta 1 interface.

The dimer-tetramer association constants of several recombinant human hemoglobins (in the CO form) have been measured by differential gel filtration. Recombinant human hemoglobin prepared from recombinant beta-chains, and mutant hemoglobins where the substitution was on the surface, beta(Thr4-->Asp), in the heme pocket, beta(Val67-->Thr), at the 2,3-DPG binding site, beta(Val1-->Met+His2del), had a twofold smaller association with respect to natural hemoglobin. In a mutant at the alpha 1 beta 2 interface, beta(Cys93-->Ala), the association constant was decreased three-fold. Conversely, in a mutant at the alpha 1 beta 1 interface, beta(Cys112-->Gly), the association constant was two- and four-fold increased with respect to natural and recombinant human hemoglobin. These differences are energetically very small, consistent with the correct folding of the recombinant hemoglobins. The stabilization of the tetrameric structure by a mutation at the alpha 1 beta 1 interface indicates that structural changes in this interface can be propagated through the protein to the alpha 1 beta 2 interface and, thereby, exert an effect on the allosteric equilibrium.

Time-resolved emission spectra of hemoglobin on the picosecond time scale.

We used front-face illumination to examine the steady-state and time-resolved emission from the intrinsic tryptophan emission of human hemoglobin (Hb). Experimental conditions were identified which eliminated all contributions of scattered light. The sensitivity obtained using front-face optics was adequate to allow measurement of the wavelength-dependent frequency response of the emission to 2 GHz. The intensity decays displayed pico- and nanosecond components in the emission at all wavelengths from 315 to 380 nm. The contribution of the picosecond component decreased from 72 to 37% over this range of wavelengths. Frequency-domain measurements were used to calculate the time-resolved emission spectra and decay-associated emission spectra. These spectra indicate that the picosecond components of the emission display maxima near 320 nm, whereas the nanosecond components are centered at longer wavelengths near 335 nm. The nanosecond components appear to be due to residual impurities which remain even in highly purified samples of Hb. However, we cannot eliminate the possibility that some of these components are due to Hb itself.

Alpha-subunit oxidation in T-state crystals of a sebacyl cross-linked human hemoglobin with unusual autoxidation properties.

In the crystal structure of human T-state hemoglobin with a sebacyl residue cross-linking the two beta-subunit Lys82,s (DecHb), the Fe atoms of the alpha-subunit hemes are found to be oxidized with a water molecule bound. The three-dimensional structure and heme geometries were compared to those of deoxyhemoglobin and other partially and fully oxidized hemoglobins [R. Liddington, Z. Derewenda, E. Dodson, R. Hubbard, G. Dodson, J. Mol. Biol. 228 (1992) 551]. The heme geometries of the alpha-subunits are consistent with those observed in oxidized structures. The proximal histidines of the alpha-subunits move toward the heme plane shifting the F-helix and FG-corner in a manner observed for partially oxidized human hemoglobin. This supports the hypothesis that these perturbations may precede the T- to R-state transition. Circular dichroism studies comparing DecHb and natural human hemoglobin in the deoxy and CO ligated forms confirm that the conformations of the deoxy forms are identical, but the ligated forms have slight differences in the solution structures. DecHb is found to be more resistant to autoxidation than natural hemoglobin. The time course of autoxidation of DecHb shows that it is virtually absent for the first 1500 min followed by a rapid increase. Thus, the discovery of the oxidation of the alpha-subunits in the deoxy-crystals is quite unexpected. The data confirm that ligation of the alpha-subunits precedes that of the beta-subunits. This may suggest a low ligand affinity of the alpha-diligated form of hemoglobin.

Surgery of the incidentally discovered mass of the adrenal gland (incidentaloma).

The authors present their experience about surgery in adrenal gland incidentaloma, during a period of 12 years (1982-1993), at the VI Division of General Surgery, University of Turin. In 17 patients, of 70 who underwent adrenalectomy, the adrenal neoplasm was located thanks to a diagnostic investigation (ECI or CT) executed for other reasons. In ten cases was diagnosed adenoma, in five cases carcinoma, in two cases cysts. In the diagnostic approach to adrenal incidentaloma we did not search for possible hormonal activity, since the patients had been previously selected from specialized endocrinological centres. At present, biological markers not being certain or absolute radiological significance so as to detect benignant from malignant forms. In accordance with international Literature the size of the neoplasm is the discriminant element for adrenalectomy. We have removed the incidentaloma in all cases, be it clinically or subclinically functional and the silent forms > 4 cm as we have observed a statistically significant difference (p < 0.0002) between the benign and malignant lesions, particularly those measuring more than 4 cm in diameter. We suggest a screening with CT scan every three months in lesions < 4 cm, silent and those > 4 cm in patients over sixty years with a morphological aspect of the bening form.

Primary hyperaldosteronism. Analysis of risk factors associated with persistent postoperative hypertension.

Between 1983 and 1992, 24 patients with primary hyperaldosteronism were operated at our division. Among these, 21 had adrenal adenoma and constituted the subjects of our study. The aim of this retrospective study was to determine risk factors that could potentially lead to persistent arterial hypertension (5 cases or 23.9%). The variables of the univariate and the multivariate analyses were: sex, age, diameter of the neoplasm, preoperative duration of the hypertension, and familial hypertension. The analysis demonstrated no statistically significant difference between the resolution of hypertension and the variables. Though not statistically significant, the only variable that approached statistical significance (p < 0.11) was familial hypertension.

[Anatomo-clinical considerations of several cases of pseudocysts of the pancreas]. / Considerazioni anatomo-cliniche su alcuni casi di pseudocisti del pancreas

The aetiology and clinical manifestations most commonly noted in pseudocysts of the pancreas are briefly described. Particular attention is given to the treatments of lesions operated under emergency conditions and the clinical aspects of pancreatic effusions sometimes encountered during the course of the disease.

[Cicatricial stenosis of colorectal anastomosis. Transanal treatment with circular stapler]. / Stenosi cicatriziale dell'anastomosi colorettale. Trattamento transanale con stapler circolare.

Anastomotic strictures complicating colorectal anastomoses can be difficult to treat. This condition must not be considered as an uncommon complication. In 20% of patients it may be a serious state that may require a therapy. Two patients treated successfully without complication with the transanal use of an CEEA stapler are presented. The staple cutter is safe and easy to use, and except for a conventional anoscope, no special equipment, including fluoroscope, is needed. On the basis of the successful results obtained, the procedure using staple cutter is recommended for the treatment of anastomotic stricture of the rectum.

[Gallbladder carcinoma. Our experience]. / Carcinoma della colecisti. Nostra esperienza.

The authors report their experience of 18 patients with primary cancer of the gallbladder. On 10 patients at stage IV, 9 had a preoperative diagnosis, while at stage 0-1 and 2 the diagnosis was intraoperative or histologic. Every patient had a cholelithiasis at the same time. The authors discuss prophylactic cholecystectomy, even without specific symptoms, and emphasize the need for a better morphological and radiomorphological classification. In the light of the new microinvasive surgical techniques, they briefly discuss laparoscopic cholecystectomy and histologic diagnosis of carcinoma of the gallbladder.

[Pelviperineal recurrences in surgery of rectal carcinoma]. / Le recidive pelvi-perineali nella chirurgia del carcinoma del retto.

A series of pelviperineal recurrences after perineal-abdominal amputation of the rectum and after anterior resection is examined. Involvement of the rectal wall, the biological behaviour of the tumour, and lymphatic diffusion represent the main causes of recurrence. In some instances, the choice of anterior resection in the treatment of middle rectum carcinoma may constitute a risk factor.

[Suspensory elements of the anal canal. Anatomo-surgical considerations]. / Elementi di sospensione del canale anale. Considerazioni anatomo-chirurgiche.

Continence depends not only on correct muscular function but also on the integrity of the suspensory structures of the anal canal, the latter being the subject of this study performed on embryological and adult specimens. The role of the perineal body as an insertion tendon common to all the striated muscles (pubo-coc-cygeus, superficial striated sphincter) which anchor the anal canal to the surrounding structures is pointed out. The characteristic behaviour of the longitudinal muscle that links the striated musculature to the visceral cylinder is also described.