Experimental demonstration of a soft x-ray self-seeded free-electron laser.

The Linac Coherent Light Source has added a self-seeding capability to the soft x-ray range using a grating monochromator system. We report the demonstration of soft x-ray self-seeding with a measured resolving power of 2000-5000, wavelength stability of 10(-4), and an increase in peak brightness by a factor of 2-5 across the photon energy range of 500-1000 eV. By avoiding the need for a monochromator at the experimental station, the self-seeded beam can deliver as much as 50-fold higher brightness to users.

Electron-transfer reactions of the reductase component of soluble methane monooxygenase from Methylococcus capsulatus (Bath).

Soluble methane monooxygenase (sMMO) catalyzes the hydroxylation of methane by dioxygen to afford methanol and water, the first step of carbon assimilation in methanotrophic bacteria. This enzyme comprises three protein components: a hydroxylase (MMOH) that contains a dinuclear nonheme iron active site; a reductase (MMOR) that facilitates electron transfer from NADH to the diiron site of MMOH; and a coupling protein (MMOB). MMOR uses a noncovalently bound FAD cofactor and a [2Fe-2S] cluster to mediate electron transfer. The gene encoding MMOR was cloned from Methylococcus capsulatus (Bath) and expressed in Escherichia coli in high yield. Purified recombinant MMOR was indistinguishable from the native protein in all aspects examined, including activity, mass, cofactor content, and EPR spectrum of the [2Fe-2S] cluster. Redox potentials for the FAD and [2Fe-2S] cofactors, determined by reductive titrations in the presence of indicator dyes, are FAD(ox/sq), -176 +/- 7 mV; FAD(sq/hq), -266 +/- 15 mV; and [2Fe-2S](ox/red), -209 +/- 14 mV. The midpoint potentials of MMOR are not altered by the addition of MMOH, MMOB, or both MMOH and MMOB. The reaction of MMOR with NADH was investigated by stopped-flow UV-visible spectroscopy, and the kinetic and spectral properties of intermediates are described. The effects of pH on the redox properties of MMOR are described and exploited in pH jump kinetic studies to measure the rate constant of 130 +/- 17 s(-)(1) for electron transfer between the FAD and [2Fe-2S] cofactors in two-electron-reduced MMOR. The thermodynamic and kinetic parameters determined significantly extend our understanding of the sMMO system.

[Chronic thallium intoxication in five German pointers of one litter]. / Chronische Thalliumvergiftung bei fünf Deutsch Drahthaar Vorstehhunden aus einem Wurf.

The difficulty of diagnosis and therapy of chronic thallium intoxication is described in five German Pointers with the same skin disease. The detection of thallium in cases of skin lesions like the cutaneous erythema with oedema and crusts or in chronic cases with multifocal alopecia is difficult. The first diagnostic information was gathered in this case from the high thallium level in the urine. The thallium concentration in the hair is subject to great variations, even in physiologic conditions. The trichogramme showed in this case pathognomonic changes like adhesion of the hair follicles. Differential diagnosis for this symmetric alopezia without pruritus are hormonal disturbances or, in puppies, the generalized form of demodicosis. The five affected dogs were treated with Fe III-Hexacyanoferrat. The clinical appearance of the skin improved slowly during a period of 1-2 months.

Component interactions in the soluble methane monooxygenase system from Methylococcus capsulatus (Bath).

The soluble methane monooxygenase system of Methylococcus capsulatus (Bath) includes three protein components: a 251-kDa non-heme dinuclear iron hydroxylase (MMOH), a 39-kDa iron-sulfur- and FAD-containing reductase (MMOR), and a 16-kDa regulatory protein (MMOB). The thermodynamic stability and kinetics of formation of complexes between oxidized MMOH and MMOB or MMOR were measured by isothermal titration calorimetry and stopped-flow fluorescence spectroscopy at temperatures ranging from 3.3 to 45 degrees C. The results, in conjunction with data from equilibrium analytical ultracentrifugation studies of MMOR and MMOB, indicate that free MMOR and MMOB exist as monomers in solution and bind MMOH with 2:1 stoichiometry. The role of component interactions in the catalytic mechanism of sMMO was investigated through simultaneous measurement of oxidase and hydroxylase activities as a function of varied protein component concentrations during steady-state turnover. The partitioning of oxidase and hydroxylase activities of sMMO is highly dependent on both the MMOR concentration and the nature of the organic substrate. In particular, NADH oxidation is significantly uncoupled from methane hydroxylation at MMOR concentrations exceeding 20% of the hydroxylase concentration but remains tightly coupled to propylene epoxidation at MMOR concentrations ranging up to the MMOH concentration. The steady-state kinetic data were fit to numerical simulations of models that include both the oxidase activities of free MMOR and of MMOH/MMOR complexes and the hydroxylase activity of MMOH/MMOB complexes. The data were well described by a model in which MMOR and MMOB bind noncompetitively at distinct interacting sites on the hydroxylase. MMOB manifests its regulatory effects by differentially accelerating intermolecular electron transfer from MMOR to MMOH containing bound substrate and product in a manner consistent with its activating and inhibitory effects on the hydroxylase.

NMR characterization of substrate binding in the phthalate dioxygenase system.

The paramagnetic enhancements in the NMR relaxation rates for the fluorine in fluorophthalates have been used to determine the position of the phthalate with respect to the mononuclear metal ion in native and metal-substituted derivatives of phthalate dioxygenase (PDO). These studies show directly that the substrate interacts with the mononuclear metal of PDO and provide the first structural characterization of this interaction. With a molecular mass of 200 kDa, PDO is one of the largest proteins studied to date by paramagnetic NMR. Two paramagnetically broadened (19)F lines were observed for monofluorophthalates bound to CoPDO. This demonstrates that fluorophthalate binds to PDO with a handedness, i.e., with the fluorine label facing to the "right" or to the "left", relative to the hyperfine tensor of the Co(II). The relative affinities of the two orientations are slightly different, with a 2-fold and 5-fold excess of the preferred orientation for 4-fluorophthalate and 3-fluorophthalate, respectively. The longitudinal relaxation rate (T(1)) and transverse relaxation rate (T(2)) data give mutually consistent fluorine to cobalt distances. These results are consistent with approximate bilateral symmetry, with the Co to 3-fluorophthalate distances ( approximately 5.5 A) approximately 25% longer than the Co to 4-fluorophthalate distances ( approximately 4. 5 A). A detailed geometric model is derived from these data. This structural characterization of the mononuclear site provides a framework to develop hypotheses for the mechanism of oxygenation by the Fe(II)-containing aromatic dioxygenases.

Structure of the soluble methane monooxygenase regulatory protein B.

The soluble methane monooxygenase (sMMO; EC 1.14.13.25) from the pseudothermophile Methylococcus capsulatus (Bath) is a three-component enzyme system that catalyzes the selective oxidation of methane to methanol. We have used NMR spectroscopy to produce a highly refined structure of MMOB, the 16-kDa regulatory protein of this system. This structure has a unique and intricate fold containing seven beta-strands forming two beta-sheets oriented perpendicular to each other and bridged by three alpha-helices. The rate and efficiency of the methane hydroxylation by sMMO depend on dynamic binding interactions of the hydroxylase with the reductase and regulatory protein components during catalysis. We have monitored by NMR the binding of MMOB to the hydroxylase in the presence and absence of the reductase. The results of these studies provide structural insight into how the regulatory protein interacts with the hydroxylase.

[Observations on the side effects after application of non-steroidal anti-inflammatory agents in dogs]. / Beobachtungen zu Nebenwirkungen nach Anwendung von nichtsteroidalen Antiphlogistika beim Hund.

This review describes with some examples possible side-effects after oral treatment with the non-steroidal anti-inflammatory drug meloxicam in dogs. Adverse effects include gastrointestinal and renal irritation such as nausea, vomiting and hemorrhagic gastroenteropathy. Therefore in case of long-term administration a dose reduction of 50 percent (single dose of 0.2 mg/kg followed by 0.1 mg/kg once daily) is recommended in dogs with chronic locomotor disorders.

Kinetic characterization of an organic radical in the ascarylose biosynthetic pathway.

The lipopolysaccharide of Yersinia pseudotuberculosis V includes a 3,6-dideoxyhexose, ascarylose, as the nonreducing end of the O-antigen tetrasaccharide. The C-3 deoxygenation of CDP-6-deoxy-L-threo-D-glycero-4-hexulose is a critical reaction in the biosynthesis of ascarylose. The first half of the reaction is a dehydration catalyzed by CDP-6-deoxy-L-threo-D-glycero-4-hexulose-3-dehydrase (E1), which is PMP-dependent and contains a redox-active [2Fe-2S] center. The second half is a reduction that requires an additional enzyme, CDP-6-deoxy-L-threo-D-glycero-4-hexulose-3-dehydrase reductase (E3, formerly known as CDP-6-deoxy-delta 3,4-glucoseen reductase), which has a FAD and a [2Fe-2S] center in the active site. Using NADH as the reductant in the coupled E1-E3 reaction, we have monitored the kinetics of a radical intermediate using both stopped-flow spectrophotometry and rapid freeze-quench EPR under aerobic and hypoxic conditions. In the EPR studies, a sharp signal at g = 2.003 was found to appear at a rate which is kinetically competent, reaching its maximum intensity at approximately 150 ms. Stopped-flow UV-vis analysis of the reaction elucidated a minimum of six optically distinguishable states in the mechanism of electron transfer from NADH to substrate. Interestingly, one of the detected intermediates has a time course nearly identical to that of the radical detected by rapid freeze-quench EPR. The difference UV-vis spectrum of this intermediate displays a maximum at 456 nm with a shoulder at 425 nm. Overall, these results are consistent with an electron transfer pathway that includes a radical intermediate with the unpaired spin localized on the substrate-cofactor complex. Evidence in support of this mechanism is presented in this report. These studies add the PMP-glucoseen radical to the growing list of mechanistically important bioorganic radical intermediates that have recently been discovered.

Kinetics of the reductive half-reaction of the iron-sulfur flavoenzyme CDP-6-deoxy-L-threo-D-glycero-4-hexulose-3-dehydrase reductase.

The conversion of CDP-4-keto-6-deoxy-D-glucose to CDP-4-keto-3,6-dideoxy-D-glucose is a key step in biosynthesis of ascarylose, the terminal dideoxyhexose of the O-antigen tetrasaccharide of the lipopolysaccharide from Yersinia pseudotuberculosis V. This transformation is catalyzed by two enzymes: CDP-6-deoxy-L-threo-D-glycero-4-hexulose-3-dehydrase (E1), which contains a pyridoxamine and a [2Fe-2S] center, and an NADH-dependent CDP-6-deoxy-L-threo-D-glycero-4-hexulose-3-dehydrase reductase (E3), which contains both an FAD and a [2Fe-2S] center. E1 reacts to form a Schiff base with CDP-4-keto-6-deoxy-D-glucose and catalyzes the elimination of the hydroxyl at position 3 of the glucose moiety, resulting in the formation of a covalently bound CDP-6-deoxy-delta(3,4)-glucoseen intermediate. E3 transfers electrons from NADH to E1, which uses these to reduce the delta(3,4)-glucoseen bond to produce CDP-4-keto-3,6-dideoxy-D-glucose. In this work, we have investigated the reductive half-reaction of E3 using both single wavelength and diode array stopped flow absorbance spectroscopy. We find that NADH binds to both oxidized (Kd = 52.5 +/- 2 microM) and two-electron-reduced (Kd = 12.1 +/- 1 microM) forms of E3. Hydride transfer from NADH to the FAD moiety occurs at 107.5 +/- 3 s-1 and exhibits a 10-fold deuterium isotope effect when (4R)-[2H]NADH is substituted for NADH. Following the hydride transfer reaction, NAD+ is released at 42.5 +/- 1 s-1 and electron transfer from the reduced FAD to the [2Fe-2S] center occurs rapidly. The extent of the intramolecular electron transfer reaction is pH-dependent with a pKa of 7.3 +/- 0.1, which may represent the ionization state of the N-1 position of the FAD hydroquinone of E3. Finally, E3 is converted to the three-electron-reduced state in a slow disproportionation reaction that consumes NADH: The [2Fe-2S] center of E3 was selectively disassembled by titration with mersalyl to give E3(apoFeS). The properties of this form of the enzyme are compared to those of the holoenzyme. Similarities and differences of the reductive half-reactions of E3 and related iron-sulfur flavoenzymes are discussed.

Active site structure of Rieske-type proteins: electron nuclear double resonance studies of isotopically labeled phthalate dioxygenase from Pseudomonas cepacia and Rieske protein from Rhodobacter capsulatus and molecular modeling studies of a Rieske center.

Continuous wave electron nuclear double resonance (CW ENDOR) spectra of [delta-15N,epsilon(-14)N]histidine-labeled phthalate dioxygenase (PDO) from Pseudomonas cepacia were recorded and found to be virtually identical to those previously recorded from [delta,epsilon-15N2]histidine-labeled protein [Gurbiel, R. J., Batie, C. J., Sivaraja, M., True, A. E., Fee, J. A., Hoffman, B. M., & Ballou, D. P. (1989) Biochemistry 28, 4861-4871]. Thus, the two histidine residues, previously shown to ligate one of the irons in the cluster [cf. Gurbiel et al. 1989)], both coordinate the metal at the N(delta) position of their imidazole rings. Pulsed ENDOR studies showed that the "remote", noncoordinating nitrogen of the histidine imidazole ring could be observed from the Rieske protein in a sample of Rhodobacter capsulatus cytochrome bc1 complex uniformly labeled with 15N but not in a sample of PDO labeled with [delta-15N,epsilon-14N]histidine, but this atom was easily observed with a sample of Rh. capsulatus cytochrome bc1 complex that had been uniformly labeled with 15N; this confirmed the conclusion from the CW ENDOR studies that ligation is exclusively via N(delta) for both ligands in the PDO center. Modifications in the algorithms previously used to simulate 14N ENDOR spectra permitted us to compute spectra without any constraints on the relative orientation of hyperfine and quadrupole tensors. This new algorithm was used to analyze current and previously published spectra, and slightly different values for the N-Fe-N angle and imidazole ring rotation angles are presented [cf. Gurbiel et al. (1989) Gurbiel, R. J., Ohnishi, T., Robertson, D. E., Daldal, F., and Hoffman, B. M. (1991) Biochemistry 30, 11579-11584]. This analysis has permitted us to refine the proposed structure of the [2Fe-2S] Rieske-type cluster and rationalize some of the properties of these novel centers. Although the spectra of cytochrome bc1 complex from Rh. capsulatus are of somewhat lower resolution than those obtained with samples of PDO, our analysis nevertheless permits the conclusion that the geometry of the cluster is essentially the same for all Rieske and Rieske-type proteins. Structural constraints inferred from the spectroscopic results permitted us to apply the principles of distance geometry to arrive at possible three-dimensional models of the active site structure of Rieske protein from Rh. capsulatus. Results from this test case indicate that similar procedures should be generally useful in metalloprotein systems. We also recorded the pulsed and CW ENDOR spectra of 57Fe-labeled PDO, and the resulting data were used to derive the full hyperfine tensors for both Fe(III) and Fe(II) ions, including their orientations relative to the g tensor. The A tensor of the ferric ion is nominally isotropic, while the A tensor of the ferrous ion is axial, having A(parallel) > A(perpendicular); both tensors are coincident with the observed g tensor, with A(parallel) of the ferrous ion lying along the maximum g-value, g1. These results were examined using refinements of existing theories of spin-coupling in [2Fe-2S]+ clusters, and it is concluded that current theories are not adequate to fully describe the experimental results.

Structure and mechanism of the iron-sulfur flavoprotein phthalate dioxygenase reductase.

Transfer of electrons between pyridine nucleotides (obligatory two-electron carriers) and hemes or [2Fe-2S] centers (obligatory one-electron carriers) is an essential step mediated by flavins in respiration, photosynthesis, and many oxygenase systems. Phthalate dioxygenase reductase (PDR), a soluble iron-sulfur flavoprotein from Pseudomonas cepacia, is a convenient model for the study of this type of electron transfer. PDR is folded into three domains; the NH2-terminal FMN binding and central NAD(H) binding domains are closely related to ferredoxin-NADP+ reductase (FNR). The COOH-terminal [2Fe-2S] domain is similar to plant ferredoxins, and can be removed by proteolysis without significantly altering the reactivity of the FNR-like domains. Kinetic studies have identified sequential steps in the reaction of PDR with NADH that involve pyridine nucleotide binding, hydride transfer to FMN, and intramolecular electron transfer from the reduced flavin to the [2Fe-2S] cluster. Crystal structures of reduced and liganded PDR correspond to some of the intermediates formed during reduction by NADH. Small structural changes that are observed in the vicinity of the cofactors upon reduction or NAD(H) binding may provide part of the reorganization energy or contribute to the gating mechanism that controls intramolecular electron transfer.

Preparation and characterization of a truncated form of phthalate dioxygenase reductase that lacks an iron-sulfur domain.

Phthalate dioxygenase reductase (PDR) is the electron transferase component of the phthalate dioxygenase system. It is a modular enzyme consisting of distinct iron-sulfur, flavin mononucleotide (FMN) and pyridine nucleotide binding domains. We have taken advantage of this modularity and removed the 10-kDa iron-sulfur domain by selective proteolytic cleavage between residues N229 and T230 in a solvent-accessible peptide that links the iron-sulfur and pyridine nucleotide binding domains. The resulting PDR(-FeS) has a molecular weight of 25,792 +/- 10 and the same amino terminus as the native PDR. It has spectral features that are very similar to the flavin component of the PDR absorbance spectrum. Remarkably, despite the magnitude of this structural modification, the kinetic, redox, and pyridine nucleotide binding properties of PDR(-FeS) are very similar to those reported for PDR [Gassner, G., et al. (1994) Biochemistry 33, 12184-12193]. The principal mechanistic feature distinguishing PDR(-FeS) from PDR is the inability of the attenuated enzyme to carry out intramolecular electron transfer. The reaction of PDR(-FeS) with NADH consists of binding and the formation of an initial Michaelis complex (MC-1') (Kd approximately 25 microM), isomerization of the enzyme (120 s-1) to form a charge-transfer complex with FMN (CT-1'), hydride transfer to the FMN (76 s-1) with formation of a second charge-transfer complex (CT*'), and finally release of nicotinamide adenine dinucleotide (NAD) (58 s-1) from the reduced enzyme. The rate of NAD release from PDR(-FeS) is nearly the same as the rate of NAD release and intramolecular electron transfer in the reductive half-reaction of PDR, which supports the idea that the release of NAD triggers intramolecular electron transfer in PDR. The midpoint potential of the oxidized/semiquinone couple of PDR(-FeS) (-170 mV) is the same as the value measured for PDR. A value of -235 mV is measured for the midpoint potential of the semiquinone/hydroquinone couple of PDR(-FeS), which is approximately 50 mV more positive than the PDR2e-/PDR3e- redox couple at pH 7. NAD binds to PDR(-FeS) about 20-fold more weakly than does NADH; the enzyme redox state has no significant influence on pyridine nucleotide binding affinity.

Reaction of phthalate dioxygenase reductase with NADH and NAD: kinetic and spectral characterization of intermediates.

Phthalate dioxygenase reductase (PDR) is an electron transferase that contains FMN, which accepts a hydride from NADH, and a [2Fe-2S] center, which transfers electrons to phthalate dioxygenase. The reduction of PDR by NADH has been studied by stopped-flow spectroscopy. Data from studies using both portio- and deuterio-NADH were analyzed by nonlinear curve fitting and numerical simulation techniques. The results of these analyses indicate that the reductive half-reaction of PDR consists of five distinct kinetic phases: (a) NADH binds to form a primary Michaelis complex (MC-1) (Kd = 50 microM). (b) The enzyme undergoes a structural change (116 +/- 5 s-1) resulting in a charge-transfer complex (CT-1). (c) The next phase in the reaction shows a deuterium isotope effect of 7.0 when (4R)-[2H]NADH (NADD) is substituted for NADH, identifying this step as the one involving hydride transfer. The rate of hydride transfer from NADH to FMN is 70 s-1, and this process results in a charge-transfer intermediate between the flavin hydroquinone anion and NAD (CT). (d) Internal electron transfer from the flavin to the iron-sulfur center, which is only 35 +/- 4 s-1, then results in an intermediate consisting of a reduced [2Fe-2S] center and a neutral flavin semiquinone (SQ). It is surprising that this rate is so slow, since the shortest interatomic distance between these centers is only 4.7 A [Correll, C. C., et al. (1992) Science 258, 1604-1610]. The 2-electron-reduced form of PDR (SQ in Figure 1) binds weakly to the reaction product, NAD (Kd = 3.7 mM), but forms a tight complex with NADH (Kd = 10 microM). (e) Two molecules of the reduced iron-sulfur flavin semiquinone (SQ) form of PDR then undergo a relatively slow second-order disproportionation reaction, resulting in one molecule of 3-electron-reduced PDR and one molecule of 1-electron-reduced PDR. The latter reacts rapidly with excess NADH to form a 3-electron-reduced PDR.

Magnetic circular dichroism studies on the mononuclear ferrous active site of phthalate dioxygenase from Pseudomonas cepacia show a change of ligation state on substrate binding.

Phthalate dioxygenase from Pseudomonas cepacia contains a mononuclear ferrous center that is strictly required for catalytic oxygen activation. The spectroscopic characterization of this iron site and its ligand interactions has been complicated in the past by interference from a Rieske-type binuclear (2Fe-2S) cluster in the enzyme, which dominates the absorption spectra and is superimposed in X-ray absorption spectra for the mononuclear site. We have used low-temperature, variable magnetic field circular dichroism spectroscopy to selectively detect the ligand field spectra of the paramagnetic mononuclear ferrous active site in the presence of the diamagnetic exchange-coupled Rieske center and observe spectral changes associated with substrate binding. The perturbations of the d-->d spectra for the mononuclear ferrous site reflect a decrease in coordination number from six to five on binding substrate. This structural change suggests that displacement of an iron ligand prepares the ferrous center for dioxygen activation.

Teichuronic acid reducing terminal N-acetylglucosamine residue linked by phosphodiester to peptidoglycan of Micrococcus luteus.

Teichuronic acid-peptidoglycan complex isolated from Micrococcus luteus cells by lysozyme digestion in osmotically stabilized medium was treated with mild acid to cleave the linkage joining teichuronic acid to peptidoglycan. This labile linkage was shown to be the phosphodiester which joins N-acetylglucosamine, the residue located at the reducing end of the teichuronic acid, through its anomeric hydroxyl group to a 6-phosphomuramic acid, a residue of the glycan strand of peptidoglycan. 31P nuclear magnetic resonance spectroscopy of the lysozyme digest of cell walls demonstrated the presence of a phosphodiester which was converted to a phosphomonoester by the conditions which released teichuronic acid from cell walls. Reduction of acid-liberated reducing end groups by NaB3H4 followed by complete acid hydrolysis yielded [3H] glucosaminitol from the true reducing end residue of teichuronic acid and [3H]glucitol from the sites of fragmentation of teichuronic acid. The amount of N-acetylglucosamine detected was approximately stoichiometric with the amount of phosphate in the complex. Partial fragmentation of teichuronic acid provides an explanation of the previous erroneous identification of the reducing end residue.

Combined proton NMR imaging and spectral analysis of locust embryonic development.

Images and spectra of desert locust [Schistocerca gregaria (Forskal)] embryos were collected in vivo using a 4.7-tesla proton ((1)H) NMR imaging spectrometer. The 100 x 100 x 500 mum image resolution and 125-nl localized spectral volumes were obtained within minutes of each other. The dynamics of embryonic development were slow enough that the time delay between imaging and spectral measurements is negligible. Thus, image and spectral data correspond closely and approximate real-time observations of embryonic changes. The above procedure was applied every 12 hr during the entire course of development. This analytical approach demonstrates that imaging and localized spectroscopy can be used to visualize and assess changes in embryonic water and lipid content in concert with the development of a living embryo.

Modification of N-methyl-N-nitrosourea-induced urinary bladder carcinogenesis in rats following stimulation of urothelial proliferation by a partial cystectomy.

The present experiments are concerned with the question whether stimulation of urothelial proliferation modifies tumor development in the urinary bladder. To induce proliferative activity of the urothelium a partial cystectomy (one-third resection of the bladder) was performed in female Wistar rats. N-Methyl-N-nitrosourea (MNU) was used as a carcinogen which acts directly on the urothelium without requiring metabolic activation. MNU was given as a single intravesicular dose of 5 mg/kg body weight via a urethral catheter. After an experimental period of 15 months rats with an intact quiescent bladder showed a tumor incidence of 32.6%. Rats having received MNU 45 h following partial cystectomy - when proliferative activity reached its peak - had developed bladder tumors with a frequency of 17.9%. Initial administration of MNU followed 24 h later by a one-third resection of the bladder resulted in a tumor incidence of only 8.8%. The histologic types of tumors induced proved to be similar to those found with other carcinogens. However, by contrast the majority of urothelial tumors were characterized by a squamous metaplasia. There was no substantial difference between the various histologic tumor types found in the resting and regenerating bladder. The mechanisms responsible for the observed inhibition of tumor development in the regenerating bladder are unknown. It is assumed that an increased capacity of the proliferating urothelial cells to repair carcinogen-induced DNA damage may play an important role.

Annulate lamellae in hemipteran preblastoderm cytoplasm and nuclei.

Annulate lamellae (AL) were found in preblastoderm cytoplasm and nuclei of the giant milkweed bug,Oncopeltus fasciatus. To the best of the authors' knowledge, this is the first report of such an occurrence among the insects. Cytoplasmic AL were scattered about the nucleus during early prophase and then accumulated during late prophase to form two dome-shaped arrays lying opposite each other with the nucleus interposed. Also, intranuclear annulate lamellae were found as single strands or vesicles within the nucleus during prophase.