A Review of the Magnetic Relaxation and Its Application to the Study of Atomic Defects in α-Iron and Its Diluted Alloys.

This review presents a comprehensive survey on intensive studies performed during the last decades on point defect reactions on α-iron (α-Fe) and its diluted alloys. Our intention is to give an actual account of the knowledge accumulated on this subject, as it has been obtained predominantly by means of the magnetic after-effect (MAE) spectroscopy. After a concise introduction into the theoretical and experimental fundamentals of this technique, the main concern is focused on the presentation and detailed discussion of the MAE spectra arising - after low-temperature electron (e-)- or neutron(n)-irradiation and subsequent annealing - in: (i) high-purity α-Fe and α-Fe doped with (ii) substitutional solutes (like Ni, V, Al, Cu, Ti, Be, Si, Mn, …) or (iii) interstitial solutes (like O, H, C, N). During the course of systematic annealing treatments, these respective spectra undergo dramatic variations at specific temperatures thereby revealing in great detail the underlying intrinsic reactions of the radiation-induced defects, i.e., reorientation, migration, clustering, dissolution and finally annihilation. In alloyed Fe systems the corresponding reaction sequences are even multiplied due to additional interactions between defects and solute atoms. Most valuable information concerning formation-, dissociation- and binding enthalpies of small, mixed clusters (of the type C i V k , N i V k ; i, k ≥ 1) has been obtained in high-purity α-Fe base material which, after charging with C or N, had been e--irradiated. Concerning the basic recovery mechanisms in α-Fe, two complementary results are obtained from the analysis of the various systems: (i) in high-purity and substitutionally alloyed α-Fe the recovery in Stage-III (200 K) is governed by a three-dimensionally migrating (H M I = 0.56 eV) stable interstitial (dumb-bell); (ii) following the formation and dissociation kinetics of small clusters (C1V k , N1V k ) in interstitially alloyed α-Fe the migration enthalpy of the monovacancy must hold the following relation H M N (0.76 eV) < H M C (0.84 eV) < H M V1. These results are in clear agreement with the so-called two-interstitial model (2IM) in α-Fe - a conclusion being further substantiated by a systematic comparison with the results obtained from nonrelaxational techniques, like i.e. positron annihilation (PA), which by their authors are preferentially interpreted in terms of the one-interstitial model (1IM).

Three-dimensional structure of ribonuclease T1 complexed with an isosteric phosphonate substrate analogue of GpU: alternate substrate binding modes and catalysis.

The X-ray crystal structure of a complex between ribonuclease T1 and guanylyl(3'-6')-6'-deoxyhomouridine (GpcU) has been determined at 2. 0 A resolution. This ligand is an isosteric analogue of the minimal RNA substrate, guanylyl(3'-5')uridine (GpU), where a methylene is substituted for the uridine 5'-oxygen atom. Two protein molecules are part of the asymmetric unit and both have a GpcU bound at the active site in the same manner. The protein-protein interface reveals an extended aromatic stack involving both guanines and three enzyme phenolic groups. A third GpcU has its guanine moiety stacked on His92 at the active site on enzyme molecule A and interacts with GpcU on molecule B in a neighboring unit via hydrogen bonding between uridine ribose 2'- and 3'-OH groups. None of the uridine moieties of the three GpcU molecules in the asymmetric unit interacts directly with the protein. GpcU-active-site interactions involve extensive hydrogen bonding of the guanine moiety at the primary recognition site and of the guanosine 2'-hydroxyl group with His40 and Glu58. On the other hand, the phosphonate group is weakly bound only by a single hydrogen bond with Tyr38, unlike ligand phosphate groups of other substrate analogues and 3'-GMP, which hydrogen-bonded with three additional active-site residues. Hydrogen bonding of the guanylyl 2'-OH group and the phosphonate moiety is essentially the same as that recently observed for a novel structure of a RNase T1-3'-GMP complex obtained immediately after in situ hydrolysis of exo-(Sp)-guanosine 2',3'-cyclophosphorothioate [Zegers et al. (1998) Nature Struct. Biol. 5, 280-283]. It is likely that GpcU at the active site represents a nonproductive binding mode for GpU [Steyaert, J., and Engleborghs (1995) Eur. J. Biochem. 233, 140-144]. The results suggest that the active site of ribonuclease T1 is adapted for optimal tight binding of both the guanylyl 2'-OH and phosphate groups (of GpU) only in the transition state for catalytic transesterification, which is stabilized by adjacent binding of the leaving nucleoside (U) group.

Upstream subsite interactions for oligonucleotide binding with ribonuclease T1.

Ribonuclease T1 (RNase T1) specifically cleaves RNA at guanylyl residues. Previous studies revealed the presence of an enzyme-subsite interaction for adenosine residues of ApGpC and ApGpU substrates (Osterman and Walz (1979) Biochemistry 10, 1984-1988). The binding of ApG and 2'-deoxyadenylyl-(3',5')-guanosine (dApG) with RNase T1 was studied in the pH range 5-9 using ultraviolet difference spectroscopy. The association constants for these dinucleoside monophosphates showed the same pH dependence both of which differed at low pH values with that for the methyl phosphoester of 5'-GMP (MepG). This difference suggested that binding of the adenosine group is strongly dependent on the deprotonation of an enzyme/ligand group with a pKa value of < or = 4.8. delta G zero for ApG binding minus that for MepG at pH > 6 yielded a delta delta G of -1.17 +/- 0.10 kcal/mol which is a measure of the contribution of the adenosine moiety to binding. ApG bound more tightly than dApG with a mean delta delta G value of -0.73 +/- 0.10 kcal/mol which demonstrated the involvement of the adenosine 2'-OH group in binding. These and other comparisons indicated that delta delta G for maximal binding the adenine base per se was -0.44 kcal/mol. delta delta G for binding pdApdG minus that for dApdG (-0.94 kcal/mol) suggested an enzyme subsite for the phosphomonoester group of former ligand.

Adult-male-specific S-warfarin (11S-OH) and progesterone (20 beta-OH) keto-reductases in rat hepatic microsomes are not identical.

Adult-male-specific reductase activities in rat hepatic microsomes use NADPH to reduce S-warfarin and progesterone to their 11S-OH and 20 beta-OH products, respectively (Apanovitch et al. (1992) Biochem. Biophys. Res. Commun. 184, 338-346). When microsomes were treated with increasing concentrations of detergent, S-warfarin (11S-OH) reductase (SW(11S)R) activity was subject to monophasic activation by Triton X-100, monophasic inhibition by sodium cholate, and, activation followed by inhibition with either CHAPS or dodecyl-beta-D-maltoside. A non-dialyzable, heat-sensitive factor in rat and rabbit sera activates microsomal SW(11S)R activity six- to eight-fold. Similar detergent inhibitions but no detergent or serum activations were observed for progesterone (20 beta-OH) reductase (P(20 beta)R) activity. A significant amount of SW(11S)R activity was lost during purification regardless of whether the detergent used for solubilization was activating or inhibiting. Octyl-Sepharose, hydroxyapatite, DEAE-cellulose and carboxymethyl matrices were used to partially purify SW(11S)R. P(20 beta)R activity co-purified with SW(11S)R and the most purified fraction contained two major and several minor polypeptides. Partially purified SW(11S)R is activated by detergents, serum, and salt. These and previous results indicate that SW(11S)R and P(20 beta)R are not identical even though they are both adult male-specific, integral membrane proteins apparently having their active sites exposed on the cytoplasmic surface of the endoplasmic reticulum.

In vitro hydrolysis of gliadin and casein peptides: secondary defect in coeliac disease shown by organ culture.

BACKGROUND: Small-intestinal organ culture was used as an in vitro model of coeliac disease, studying biopsy specimens from patients with coeliac disease, cow's milk allergy, and controls. METHODS: Organ culture incubations were done using the pure gliadin peptide B3144 (amino acid sequences 3-56 of alpha-type gliadins) and a control peptide from casein (amino acid sequences 152-193 of alpha s1-casein). The importance of using negative controls was stressed by non-specific tissue damage. By reversed-phase high-performance liquid chromatography of organ culture supernatants, 27 specimens were further investigated. RESULTS: There was good retrieval of peptide calibration peaks after culture. Qualitative and quantitative evaluation of chromatography runs showed degradation of at least 29% of B3144 and 37% of Cas-P. Normal mucosa (controls and coeliac patients on a gluten-free diet) was able to hydrolyse peptide fractions completely, whereas incubation with damaged mucosa (coeliac disease, cow's milk allergy) left initial peptides. CONCLUSION: It is concluded, using a pure single gliadin peptide, that deficient peptide hydrolysis in coeliac disease was a secondary event.

[Severe perforating eye injury caused by an air bag in a traffic skid accident]. / Schwere perforierende Augenverletzung durch Airbag bei Schleuderunfall.

BACKGROUND: After a penetrating injury of the eye following a car accident the driver demanded accurate investigations by the Institute for Legal Medicine of Zurich concerning the security of the equipment, especially the airbag. This led to an astonishing explication of the mechanism of the injury, not without consequences for safety measures for drivers and front seat passengers in air bag equipped cars. CASE REPORT: Due to a relatively harmless accident, the driver suffered from a severe penetrating injury of the right eye after the airbag deployed. The front seat passenger, having the seat belts fastened, was not injured. The accident was investigated by the Institute of Legal Medicine of Zurich. RESULTS: The analysis of the accident showed that the airbag had deployed properly. The cover of the airbag showed no defects of substance. With the precise examination of the interior of the car a broken tobacco pipe came to light. CONCLUSIONS: In this case not the airbag itself but a tobacco pipe held in the hand by the driver during the airbag ignition caused a severe injury of the eye. This case report illustrates the hazard of having any rigid object between the occupant and the deploying air bag. In conclusion, the drivers and front seat passengers of an airbag equipped car can only profit from the considerable security gain, if they know about these risks and adapt their behaviours to the new surroundings, but also the car manufactures have to instruct the customers properly.

Airbag deployment and eye perforation by a tobacco pipe.

Airbags have been shown to reduce injuries and save the lives of car occupants in a crash. Like any protection system, airbags potentially introduce some new risks if no appropriate countermeasures are taken. A case of a relatively moderate frontal impact is described where the driver of an airbag-equipped car suffered a severe penetrating eye injury after the airbag deployed. Since the airbag fabric itself was excluded as an injury-producing structure, other objects such as eyeglasses, a wrist-watch, a bracelet, and a large finger ring had to be assessed. The investigation of the car interior as well as the morphologic details of the injuries to the eye and the face revealed that the most likely candidate for the injury was a tobacco pipe, which was probably being held in one hand and was broken apart by the deploying airbag and projected into the face of the driver. This case illustrates the hazard of having any rigid object between the occupant and the deploying airbag. The desirability of warning car occupants of the potential hazards which can result from today's protection systems is also discussed.

Genetic and developmental diversity of hepatic cytochromes P450. Warfarin and progesterone metabolism by hepatic microsomes from four inbred strains of rat.

Hepatic microsomes from immature and sexually mature male and female ACI/SEGHsd, F344/NHsd, SHR/NHsd, and WKY/NCrl inbred rats were used to study cytochrome P450 (P450)-catalyzed oxidations of progesterone and both enantiomers of warfarin. Strains were selected on the basis of their different, homozygous allelic compositions for CYP2C11 and CYP2C13 (Rampersaud and Walz, 1992), but no correlations with the microsomal activities were observed. However, correlations were made regarding catalytic activities and the developmental control of CYP2A1 and CYP2C11 levels in microsomes. Other correlations were found for reactions of both warfarin enantiomers at the same atom or for a given enantiomer at different positions, and these appear to involve several P450 isozymes. Strain-dependent activity differences mainly involved the SHR/NHsd and WKY/NCrl strains. WKY/NCrl rats were the most unique strain, having low levels of CYP2C11 in adult males compared with the other inbreds but relatively high S-warfarin 4'- and 6-hydroxylase activities in immature animals of both sexes and adult females. These results suggest that the regulation and/or allozymic composition of hepatic P450s are different for WKY/NCrl rats, which makes them a poor choice as "normotensive controls" in comparison with hypertensive SHR rats.

Exocyclic-keto reductase activities for progesterone and S-warfarin in hepatic microsomes from adult male rats.

Hepatic microsomes from adult male rats representing six inbred strains catalyzed quantitatively significant, NADPH dependent reductions of progesterone to the 20 beta (20R) alcohol and S-warfarin to its 11S-OH product. Microsomes from mature females and immature rats of both sexes were essentially devoid of these activities. Two strains of rat evidenced about 21% of these activities compared with the other strains and both activities were 25-81% repressed by treatment of rats with phenobarbital (PB). An excellent linear correlation was demonstrated for the two activities considering sex, age, NADPH much greater than NADH preference, PB-repression and strain differences. However, detergent latency (71%) and resistance to trypsinolysis were only observed for the keto-reductase activity with S-warfarin. Microsomes also catalyzed the reduction of progesterone to its 20 alpha-OH derivative but this activity preferred NADH greater than NADPH, was induced 2.7-fold by PB and was essentially independent of age, sex and animal strain. Furthermore, unlike the 20 beta-OH activity, this reduction was resistant to proteolytic inactivation.

Safety and economic aspects of continuous mammalian cell culture.

Mammalian cell cultures are the most appropriate host cells for recombinant DNA derived products if complex protein structures have to be synthesized in their native form. Due to their physiological behaviour they grow either adherent or in suspension. For the attachment of adherent cells, microcarriers or wire springs can be applied to increase the internal surface of the bioreactor. Both systems provide a simplified media exchange but, however, show some limitations in scale up. In contrast, suspension culture systems as homogeneous systems independent of any carrier have not shown any limitation in scale up. Because most cell lines which are of commercial interest grow in suspension, this technology is best advanced and used in batch and continuous mode. Although mammalian cell cultures are sensitive to hydrodynamic shear forces, technologies for deep tank production are developed which allow stirrer tip speed of up to 1.5 m s-1 sufficient for oxygen uptake, suspension of cells and homogeneous supply with nutrients. For long term bioprocesses without selection pressure it has to be considered that transformed cell lines might show genetic instability due to their variations of chromosomes. In addition, sterile technology becomes an important factor in long term bioprocesses. The decision as to which cell culture system should be chosen, whether batch or continuous processes should be applied essentially is based on the capital investment, the amount of material to be produced, genetic stability of the production cell line, reliability of sterile technology and the flexibility required in the production plant. Under the assumption that 20 kg of a protein have to be produced per year and the same product concentrations in the harvest fluid are reached in the batch process and for instance in the chemostat, it can be considered that the capital investment for one 10,000 l batch process and a 2 x 2,000 l continuous process, necessary to produce the amount of material, is comparable. Risk of microbial contamination or technical failure can be considered to be fairly low in the batch process. The economic risk for long term bioprocess in the chemostat can be expected to be medium and high in the perfusion system which is in the large scale not technically fully satisfactory. In addition, due to the longer down time period after contaminations and the start up of the continuous process, the annual yield of the batch process can be considered to be higher.(ABSTRACT TRUNCATED AT 400 WORDS)

Cytochrome P-450 polypeptides in pulmonary microsomes from rats.

Pulmonary microsomal polypeptides from different strains of rats were resolved using two-dimensional electrophoresis and were further characterized by in situ peptide mapping. Triton X-114 detergent separation was used to enrich cytochromes P-450 (P-450) and other integral membrane proteins from pulmonary microsomes, and these were directly compared with corresponding polypeptides from hepatic microsomes. The results demonstrated that P-450b and epoxide hydrolase were present in the lungs of male and female rats and that their expression in this tissue was independent of phenobarbital treatment. P-450e, which is co-induced with P-450b in the liver, was not detected in pulmonary microsomes under any condition. Four other pulmonary microsomal polypeptides were characterized and preliminary evidence suggested that they represent unique isozymic forms of P-450 with three of them being related to P-450b.

At least six forms of extremely homologous cytochromes P-450 in rat liver are encoded at two closely linked genetic loci.

A subpopulation of phenobarbital-induced cytochrome P-450 in rat liver has been shown to consist of four closely related forms of the enzyme that appeared to be strain-related. In the present study, polypeptides composing this family were analyzed by two-dimensional electrophoresis of hepatic microsomes from 64 individual phenobarbital-treated rats. The animals surveyed included both sexes from four inbred and five outbred strains/colonies and F1 progenies from 10 crosses. Two new members of this polypeptide family were identified on the basis of their unique electrophoretic behavior and peptide maps. Eight phenotypes were observed that consisted of two to four member polypeptides. The six closely related cytochromes P-450 were found to be encoded at two genetic loci with at least four alleles at the P-450b locus and at least two alleles at the P-450e locus. Most colonies of outbred strains were characterized by polymorphism at one or both of these loci, and in no case did they contain unique alleles. Analyses of parents and their F1 progenies indicated that the P-450b and P-450e loci are closely linked on the same autosome and are expressed codominantly. Furthermore, the products of these loci appear to be coordinately regulated. The extreme homology between P-450b and P-450e genes, their high degree of polymorphism, and their close linkage suggest that they are subject to the same genetic mechanisms that maintain these features in other multigene families.

Species differences in cytochromes P-450 and epoxide hydrolase: comparisons of xenobiotic-induced hepatic microsomal polypeptides in hamsters and rats.

Cytochromes P-450 and epoxide hydrolase in hamsters were studied by using two-dimensional gel electrophoresis of hepatic microsomes from untreated animals and those treated with phenobarbital, 3-methylcholanthrene, beta-naphthoflavone, trans-stilbene oxide, and pregnenolone-16 alpha-carbonitrile. Coelectrophoresis with corresponding microsomes from rats and in situ peptide mapping were used to identify resolved microsomal polypeptides as cytochromes P-450 or epoxide hydrolase. Two forms of hepatic microsomal epoxide hydrolase were shown to exist in hamsters; these evidenced extensive structural homology with the corresponding enzyme in rats and were induced by the same xenobiotics. At least eight inducible polypeptides in microsomes from hamsters were tentatively identified as cytochromes P-450. Two of these were electrophoretically identical and structurally related with previously characterized forms of the enzyme in rats. Homologues of several major cytochromes P-450 induced by pregnenolone-16 alpha-carbonitrile and/or phenobarbital in the rat were apparently not present in the hamster. In most cases, putative forms of inducible cytochrome P-450 in the hamster existed at significant levels in microsomes from untreated animals whereas in rats the levels of most inducible forms of the enzyme were low in control microsomes, being more strictly dependent on xenobiotic pretreatment. In contrast with epoxide hydrolase, the molecular complexity of hepatic cytochrome P-450 seems to be comparable for rats and hamsters, but the structure and control of these hemoproteins appear to have markedly diverged.

Polypeptide patterns of hepatic microsomes from Long-Evans rats treated with different xenobiotics.

Two-dimensional gel electrophoresis was used to analyze hepatic microsomal polypeptides after treatment of immature, male Long-Evans rats with 3-methylcholanthrene, pregnenolone-16 alpha-carbonitrile, isosafrole, SKF-525A, Aroclor-1254, gamma-chlordane, or trans-stilbene oxide. Epoxide hydrolase and cytochromes P-450a, P-450bLE, P-450c, P-450d, and P-450e were all identified as resolved polypeptides in these electrophoretograms. Idiosyncratic polypeptide patterns characterized the microsomal preparations following treatment of rats with each inducing agent. Immunochemically identical cytochromes P-450bLE and P-450e were always present at the same relative levels even though their total amount varied 3-fold after induction by isosafrole, SKF-525A, Aroclor-1254, gamma-chlordane, and trans-stilbene oxide. Cytochromes P-450c and P-450d were coinduced by 3-methylcholanthrene, isosafrole, and Aroclor-1254, but their relative amounts varied. Pregnenolone-16 alpha-carbonitrile treatment resulted in an increase of a single major microsomal polypeptide which was also induced by phenobarbital, isosafrole, SKF-525A, Aroclor-1254, and trans-stilbene oxide. Only one polypeptide was identified as epoxide hydrolase in all of the microsomes analyzed. The results suggest that the levels of cytochromes P-450bLE and P-450e may be subject to coordinate control, whereas the other cytochromes P-450 are independently regulated.

Multiple, immunoidentical forms of phenobarbital-induced rat liver cytochromes P-450 are encoded by different mRNAs.

It was shown previously that four immunochemically identical forms of phenobarbital-induced hepatic cytochrome P-450 exist in unique combinations which characterize different strains and colonies of rats (Vlasuk, G. P., Ghrayeb, J., Ryan, D. E., Reik, L., Thomas, P. E., Levin, W., and Walz, F. G., Jr. (1982) Biochemistry 21, 789-798). One colony of Long-Evans rats exhibited only cytochromes P-450bLE and P-450e among these immunorelated enzymes; whereas, one colony of Holtzman rats was characterized by cytochromes P-450bH and P-450e. After phenobarbital treatment, hepatic poly(A)+-mRNA was isolated from these groups of rats and translated in vitro. The 35S-labeled products were immunoisolated with antibody to cytochrome P-450bLE and analyzed by two-dimensional gel electrophoresis. The results indicated that the products synthesized in vitro correspond exactly to the two particular forms of the enzyme that characterize liver microsomes from each of these groups of rats. It is concluded that different structural genes encode these immunorelated forms of cytochrome P-450 and that significant post-translational processing of their polypeptide products does not occur in vivo.

Multiplicity strain differences, and topology of phenobarbital-induced cytochromes P-450 in rat liver microsomes.

The multiplicity of phenobarbital-induced cytochromes P-450 in live microsomes from male rats was investigated by using two-dimensional gel electrophoresis, peptide fingerprinting, and immunoaffinity chromatography. Two colonies each of Holtzman and Long-Evans rats were studied. Four molecular forms of phenobarbital-induced cytochromes P-450 were distinguished as polypeptides (designated PB3, variant PB3, PB4, and PB5) which showed apparent immunochemical identity and greater than or equal to 95% fingerprint homology. Two of these polypeptides corresponded to cytochrome P-450b [Ryan, D., Thomas, P. E., Korzeniowski, D., & Levin, W. (1979) J. Biol. Chem. 254, 1365-1374] and cytochrome P-450e [Ryan, D., & Levin, W. (1981) Fed. Proc., Fed. Am. Soc. Exp. Biol. 40, 1640] which had been purified from Long-Evans rats (variant PB3 and PB5, respectively). Each rat colony was characterized by unique combinations of two or three of these immunochemically related forms of cytochromes P-450. Cytochrome P-450e was present in rats from all four colonies, but cytochrome P-450b was only found in Long-Evans rats. Polypeptide PB3 was only found in the two colonies of Holtzman rats, whereas polypeptide PB4 was present in one colony each of Holtzman and Long-Evans rats. In addition to these forms of cytochrome P-450, rats from each colony also evidenced three other major phenobarbital-induced polypeptides which gave unique fingerprints, and one of these was identified as representing epoxide hydrolase. Proteolytic digestion studies of intact microsomes demonstrated that the four immunochemically identical forms of cytochrome P-450 were partially exposed on the outer (cytoplasmic) surface of microsomes. However, polypeptide PB3 was characterized by the greatest rate of proteolytic degradation. These results clearly demonstrate that phenobarbital-induced cytochromes P-450 include microheterogeneous proteins which show remarkable variations related to rat strains and/or colony.