Prognostic role of vascular endothelial growth factor in hepatocellular carcinoma: systematic review and meta-analysis.

Hepatocellular carcinoma (HCC) is a highly vascular tumour that expresses vascular endothelial growth factor (VEGF). Various studies have evaluated the prognostic value of VEGF levels in HCC. Its overall test performance remains unclear, however. The aim was to perform a systematic review and meta-analysis of prognostic cohort studies evaluating the use of VEGF as a predictor of survival in patients with treated HCC. Eligible studies were identified through multiple search strategies. Studies were assessed for quality using the Newcastle-Ottawa Tool. Data were collected comparing disease-free and overall survival in patients with high VEGF levels as compared to those with low levels. Studies were pooled and summary hazard ratios were calculated. A total of 16 studies were included for meta-analysis (8 for tissue and 8 for serum). Methodological analysis indicated a trend for higher study quality with serum studies as compared to tissue-based investigations. Four distinct groups were pooled for analysis: tissue overall survival (n=251), tissue disease-free survival (n=413), serum overall survival (n=579), and serum disease-free survival (n=439). High tissue VEGF levels predicted poor overall (HR=2.15, 95% CI: 1.26-3.68) and disease-free (HR=1.69, 95% CI: 1.23-2.33) survival. Similarly, high serum VEGF levels predicted poor overall (HR=2.35, 95% CI: 1.80-3.07) and disease-free (HR=2.36, 95% CI 1.76-3.16) survival. A high degree of inter-study consistency was present in three of four groups analysed. Tissue and serum VEGF levels appear to have significant predictive ability for estimating overall survival in HCC and may be useful for defining prognosis in HCC.

Abnormal nonshivering thermogenesis in mice with inherited defects of fatty acid oxidation.

When placed in the cold (4 degreesC), BALB/cByJ mice of both genders rapidly lose body temperature as compared with the control strain, C57BL/6J. This sensitivity to cold resembles that previously described for mice with a defect in nonshivering thermogenesis due to the targeted inactivation of the brown adipocyte-specific mitochondrial uncoupling protein gene, Ucp1. Genetic mapping of the trait placed the gene on chromosome 5 near Acads, a gene encoding the short chain acyl CoA dehydrogenase, which is mutated in BALB/cByJ mice. The analysis of candidate genes in the region indicated a defect only in the expression of Acads. Confirmation of the importance of fatty acid oxidation to thermogenesis came from our finding that mice carrying the targeted inactivation of the long chain acyl CoA dehydrogenase gene (Acadl) are also sensitive to the cold. Both of these mutations attenuate the induction of genes normally responsive to adrenergic signaling in brown adipocytes. These results suggest that the action of fatty acids as regulators of gene expression has been perturbed in the mutant mice. From a clinical perspective, it is important to determine whether defects in thermogenesis may be a phenotype in human neonates with inherited deficiencies in fatty acid beta-oxidation.

Cytochrome b5 and NADH-cytochrome-b5 reductase from sipunculan erythrocytes; a methemerythrin reduction system from Phascolopsis gouldii.

We report the purification and characterization of a soluble cytochrome b5 from coelomic erythrocytes of the sipunculan worm, Phascolopsis gouldii. We also report the isolation and purification of a membrane-bound NADH-cytochrome-b5 reductase from these erythrocytes. The non-heme iron protein, hemerythrin (Hr), is known to be the oxygen carrier in these erythrocytes. The aforementioned purified cytochrome b5 and reductase together catalyze the reduction of P. gouldii [Fe(III),Fe(III)]metHr to [Fe(II),Fe(II)deoxyHr by NADH. EPR spectroscopy demonstrates that a redox process involving formation of the intermediate [Fe(II),Fe(III)]semi-metHr occurs within intact sipunculan erythrocytes as well as in the system of purified components. The rhombic g-tensor of the EPR signal in both cases resembles that of (semi-met)RHr, the form obtained by one-electron reduction of metHr. These observations suggest that cytochrome b5 and NADH-cytochrome-b5 reductase in sipunculan erythrocytes function to counteract autoxidation of oxyHr. The sequence of electron flow in the system of purified components is: NADH----NADH-cytochrome-b5 reductase----cytochrome b5----metHr. At pH 7.5, the reduction of metHr in this system occurs in two phases, only the first of which is dependent on concentration of cytochrome b5. From an analysis of the kinetics and the EPR time-course, we propose that the two phases represent sequential reduction of met- to semi-metHr and reduction of semi-metHr to deoxyHr. This report represents the first demonstration of a physiological system for reduction of metHr.

Purification and properties of a membrane-bound NADH-cytochrome-b5 reductase from erythrocytes of the sipunculid worm, Phascolopsis gouldii.

The purification to homogeneity of the membrane-bound NADH-cytochrome-b5 reductase from erythrocytes of the sipunculid, Phascolopsis gouldii is reported. This highly purified reductase has allowed more detailed characterizations of its molecular and kinetic properties than was possible in a previous study (Utecht, R.E. and Kurtz, D.M., Jr. (1988) Biochim. Biophys. Acta 953, 164-178). The reductase has a molecular weight of 34,000 and contains FAD as the prosthetic group. In aqueous solution containing 0.5 vol% Triton X-100, the reductase forms an aggregate of Mr approximately 220,000. A higher purity preparation of P. gouldii erythrocyte b5 was also obtained. The combination of purified, solubilized reductase and cytochrome b5 was shown to catalyze the quantitative two-electron reduction of [Fe(III),Fe(III)]methemerythrin to [Fe(II),Fe(II)]deoxyhemerythrin by NADH. The P. gouldii NADH-cytochrome b5 reductase is the first from hemerythrin-containing erythrocytes to be purified and characterized. This methemerythrin reduction system appears to be analogous to methemoglobin reductases from vertebrate erythrocytes.

Conversion of non-functional to functional iron following reconstitution of hemerythrin.

A recent report from this laboratory (Zhang, J.-H., Kurtz, D.M., Jr., Xia, Y.-M. and Debrunner, P.G. (1991) Biochemistry 30, 583-589) described a procedure for reconstitution of a functional di-iron site in the octameric, non-heme iron O2-carrying protein, hemerythrin by addition of ferrous salts to apoprotein, followed by slow dilution of the denaturant. Although the resulting protein contained its full complement of iron, i.e., 2 Fe per subunit, about 30% of the iron was found to remain ferrous under ambient O2, i.e., this iron was incapable of forming an O2 adduct. In this report a method is described for obtaining essentially fully functional hemerythrin by passage of the freshly reconstituted protein through an [oxy/30% non-functional----met----deoxy----oxy redox cycle. UV/vis absorption and 57Fe Mössbauer spectroscopies show that little or no non-functional iron remains in the reconstituted oxyhemerythrin after the redox cycle. Quantitations of protein and diiron sites show that, during the first step of the redox cycle, the non-functional iron is converted to a form that is spectroscopically indistinguishable from that of native methemerythrin. Far-UV circular dichroism shows that the secondary structure of this reconstituted methemerythrin is essentially identical to that of native protein. Non-denaturing polyacrylamide gel electrophoresis shows that the size and charge of the native and reconstituted proteins before and after redox cycling are essentially identical. These results indicate that the non-functional iron is converted to a functional form by the redox cycling, and that the key step in this conversion is the [oxy/30% non-functional]----met transformation.

Amino-acid sequences of the alpha- and beta-subunits of hemerythrin from Lingula reevii.

The complete amino-acid sequences of the alpha- and beta-subunits of hemerythrin from the brachiopod Lingula reevii have been determined. These subunits are found in equal proportions in coelomic hemerythrocytes and form an octamer, presumably with an alpha 4 beta 4 composition. Both subunits were found to consist of 117 residues and to show 65% sequence identity to each other. Sequences of the alpha- and beta-subunits of L. reevii hemerythrin are closely related to their counterparts in hemerythrin from the related brachiopod, Lingula unguis, showing 95% and 87% sequence identities, respectively. Sequence alignments show that 25 residues in the lingulid hemerythrin subunits are identical to those found in other hemerythrins and myohemerythrins. These conserved residues include those known to provide iron ligands. However, these comparisons also indicate that the lingulid hemerythrin sequences are distinct from those of the sipunculid and annelid hemerythrins and myohemerythrins.

Myohemerythrin from the sipunculid, Phascolopsis gouldii: purification, properties and amino acid sequence.

Two previously unknown isoforms, labelled iso I and iso II, of the oxygen-carrying protein, myohemerythrin, have been isolated from carcasses of the sipunculid worm, Phascolopsis gouldii. The two isoforms have non-identical N-terminal amino acid sequences and slightly different absorption spectra in the met form. Far-ultraviolet circular dichroism shows that iso I contains approximately 69% alpha-helix. The complete amino acid sequence for iso I was obtained. The molecular weight calculated from this amino acid sequence and including the active site Fe-O-Fe unit, is 13,829. All of the physical and chemical properties of iso I noted above, including the amino acid sequence, are very similar to those of T. zostericola myohemerythrin. Except for the amino acid sequence, these properties are also very similar to that of a subunit in hemerythrin, the octameric analog found in hemerythrocytes. Only 58 of the 113 residues in P. gouldii hemerythrin are conserved in iso I. Sequence comparisons were used to help identify residues responsible for maintaining the common tertiary and diiron site structures in hemerythrin and myohemerythrin. The seven iron ligand residues previously identified in crystal structures of hemerythrin and myohemerythrin are conserved in iso I. However, none of the ten residue pairs previously identified as engaging in direct salt-bridge or hydrogen bond interactions between subunits in the hemerythrin octamer are conserved in iso I.

Oxygen-carrying proteins: three solutions to a common problem.

Nature has used transition-metal ions with unpaired d-electrons to overcome the kinetic inertness of O2 and to control its thermodynamic tendency towards reduction. High-resolution X-ray crystal structures of O2-carrying proteins show that Nature has devised three distinct solutions to the problem of reversible O2 binding. The three types can be classified according to their active sites: Hb (haem iron); Hr (non-haem di-iron); and Hcy (dicopper). The reversible O2 binding to the three types of active site are formally oxidative additions: Fe(II) to Fe(III)-O2- for Hb; [Fe(II),Fe(II)] to [Fe(III),Fe(III)O(2)2-] for Hr; and [Cu(I),Cu(I)] to [Cu(II)(mu-O(2)2-) Cu(II)] for Hcy. In all cases the O-O bond is weakened, but not cleaved, upon binding. The 'textbook' explanation for discrimination against CO and O2 binding to Hb has been revised: steric constraints to the preferred linear Fe-C-O geometry imposed by the 'distal' histidine are no longer thought to play a major role. Instead, recent experimental evidence indicates that the polarity of the binding pocket favours the polar Fe-O-O unit over the relatively non-polar Fe-C-O unit, and that a C-O-binding pocket near the haem also inhibits the preferred linear Fe-C-O geometry. Reversible O2 binding to the di-iron site of Hr involves an internal proton transfer as well as electron transfer to O2, but the elementary steps governing the rates of O2 binding and release, especially the effects of the surrounding protein, remain to be delineated. An unusual side-on-bonded O2 that bridges the two copper ions explains both the unusually low O-O stretching frequency and the diamagnetism of oxyHcy. O2-activating-enzyme counterparts exist for each of the three known types of O2-carrying protein. Detailed comparisons of these protein/enzyme pairs are likely to clarify the factors that tune the delicate balance between reversible O2 binding and controlled O-O bond cleavage.

Thermal stability of Clostridium pasteurianum rubredoxin: deconvoluting the contributions of the metal site and the protein.

To provide a framework for understanding the hyperthermostability of some rubredoxins, a comprehensive analysis of the thermally induced denaturation of rubredoxin (Rd) from the mesophile, Clostridium pasteurianum was undertaken. Rds with three different metals in its M(SCys)4 site (M = Fe3+/2+, Zn2+, or Cd2+) were examined. Kinetics of metal ion release were monitored anaerobically at several fixed temperatures between 40 and 100 degrees C, and during progressive heating of the iron-containing protein. Both methods gave a thermal stability of metal binding in the order Fe2+ << Fe3+ < Zn2+ < Cd2+. The temperature at which half of the iron was released from the protein in temperature ramp experiments was 69 degrees C for Fe2+ Rd and 83 degrees C for Fe3+ Rd. Temperature-dependent changes in the protein structure were monitored by differential scanning calorimetry, tryptophan fluorescence, binding of a fluorescent hydrophobic probe, and 1H NMR. Major but reversible structural changes, consisting of swelling of the hydrophobic core and opening of a loop region, were found to occur at temperatures (50-70 degrees C) much lower than those required for loss of the metal ion. For the three divalent metal ions, the results suggest that the onset of the reversible, lower-temperature structural changes is dependent on the size of the MS4 site, whereas the final, irreversible loss of metal ion is dependent on the inherent M-SCys bond strength. In the case of Fe3+ Rd, stoichiometric Fe3+/cysteine-ligand redox chemistry also occurs during metal ion loss. The results indicate that thermally induced unfolding of the native Cp Rd must surmount a significant kinetic barrier caused by stabilizing interactions both within the protein and within the M(SCys)4 site.

2D 1H and 3D 1H-15N NMR of zinc-rubredoxins: contributions of the beta-sheet to thermostability.

Based on 2D 1H-1H and 2D and 3D 1H-15N NMR spectroscopies, complete 1H NMR assignments are reported for zinc-containing Clostridium pasteurianum rubredoxin (Cp ZnRd). Complete 1H NMR assignments are also reported for a mutated Cp ZnRd, in which residues near the N-terminus, namely, Met 1, Lys 2, and Pro 15, have been changed to their counterparts, (-), Ala and Glu, respectively, in rubredoxin from the hyperthermophilic archaeon, Pyrococcus furiosus (Pf Rd). The secondary structure of both wild-type and mutated Cp ZnRds, as determined by NMR methods, is essentially the same. However, the NMR data indicate an extension of the three-stranded beta-sheet in the mutated Cp ZnRd to include the N-terminal Ala residue and Glu 15, as occurs in Pf Rd. The mutated Cp Rd also shows more intense NOE cross peaks, indicating stronger interactions between the strands of the beta-sheet and, in fact, throughout the mutated Rd. However, these stronger interactions do not lead to any significant increase in thermostability, and both the mutated and wild-type Cp Rds are much less thermostable than Pf Rd. These correlations strongly suggest that, contrary to a previous proposal [Blake PR et al., 1992, Protein Sci 1:1508-1521], the thermostabilization mechanism of Pf Rd is not dominated by a unique set of hydrogen bonds or electrostatic interactions involving the N-terminal strand of the beta-sheet. The NMR results also suggest that an overall tighter protein structure does not necessarily lead to increased thermostability.

Structure and chromosomal location of the mouse medium-chain acyl-CoA dehydrogenase-encoding gene and its promoter.

Medium-chain acyl-coenzyme A dehydrogenase (MCAD; mouse gene Acadm; human gene ACADM) catalyzes the initial step of fatty acid beta-oxidation in mitochondria. Inherited MCAD deficiency is an autosomal recessive disorder that occurs at high frequency in humans and is associated with considerable morbidity and mortality. We have cloned and characterized mouse Acadm which spans approximately 25 kb and contains 12 exons. The promoter region does not contain TATA or CAAT boxes and is G + C-rich (60%) within 200 bp of the cap site. A CpG island extends from 5' of the transcription start point into intron 1. The 5' regulatory region and a portion of intron 1 contain several Sp1 consensus sites and three regions containing hexamer DNA sequences that match the binding consensus for steroid/thyroid nuclear receptors. These putative nuclear receptor response elements (NRRE) share DNA sequence homology and electrophoretic mobility shift characteristics with known NRRE in the human ACADM promoter [Carter et al., J. Biol. Chem. 268 (1993) 13805-13810]. We have mapped mouse Acadm to the distal end of chromosome 3. Sequences previously localized to chromosome 8 are shown to be a pseudogene, and an additional pseudogene was identified on chromosome 11.

Immunoblastic sarcoma.

A systemic, malignant disorder of immunoblasts has been reported to occur as a late transition phase of immunoblastic lymphadenopathy in an older population. With a background of polyclonal gammopathy, drug sensitivity, lupus erythematosus or rheumatoid arthritis, there may develop a progressive "cloning out" of monomorphous immunoblasts to form immunoblastic sarcoma. Recently, a 17-year-old, postpartum girl without these bizarre hypersensitivity phenomena developed a rapidly fatal, systemically malignant process with severe clinical complications. No historical incitent was identified. The pregnancy had been uneventful and birth had produced a thriving, viable infant. This case may represent the youngest patient with primary, malignant immunoblastic disease occurring in the absence of a "hypersensitivity" state. A brief discussion of the clinical situation, management, and autopsy findings, is presented. Basic morphologic criteria for proper differentiation of malignant immunoblastic problems, lymphoma, and Hodgkin's disease are discussed.

Hodgkin's disease of the endometrium.

We report a case of Hodgkin's disease of the endometrium. The endometrial stroma is replaced by a polymorphic cellular infiltrate in which Reed-Sternberg cells are seen. The patient also has a history of stage IV-B Hodgkin's disease that was diagnosed and treated three years earlier. To our knowledge, Hodgkin's disease of the endometrium has not been described.

31P NMR probes of sipunculan erythrocytes containing the O2-carrying protein hemerythrin.

Reported are the first examinations by 31P NMR of erythrocytes containing the non-heme iron O2-carrying protein hemerythrin (Hr). Intact coelomic erythrocytes from the sipunculids Phascolopsis gouldii and Themiste zostericola were shown by 31P NMR to contain O-phosphorylethanolamine and 2-aminoethylphosphonate as the major soluble phosphorus metabolites. This combination of major metabolites appears to be unique to sipunculan erythrocytes. Nucleoside triphosphates and mannose 1-phosphate were present in lower concentrations. The concentration of O-phosphorylethanolamine within P. gouldii erythrocytes was established to be greater than 20 mM. T. zostericola erythrocytes contained relatively high levels of 2-aminoethylphosphonate (on the order of 0.1 M) and lower levels of O-phosphorylethanolamine compared with those of P. gouldii. For P. gouldii and T. zostericola the intracellular pHs were determined to be 7.2 +/- 0.1 and 7.1 +/- 0.1, respectively, in air-equilibrated erythrocytes, and 6.5 +/- 0.1 in anaerobic P. gouldii erythrocytes. O-Phosphorylethanolamine was found to bind weakly to P. gouldii metHr (Kf approximately 7 M-1). This interaction is best characterized by either negative cooperativity or nonspecific binding. O-Phosphorylethanolamine strongly inhibits azide binding to the iron site of P. gouldii metHr at pH 7.2. The rate of azide binding decreases by approximately 85-fold in the presence of 0.33 M O-phosphorylethanolamine. However, neither O-phosphorylethanolamine nor 2-aminoethylphosphonate at 0.33 M was found to have any significant effect on O2 affinity of P. gouldii deoxyHr. Alternative functions for the two metabolites are suggested.

Chromatographic separation of extruded iron-sulfur cores from the apoproteins of Clostridium pasteurianum and spinach ferredoxins in aqueous Triton X-100/urea.

Iron-sulfur core extrusions from spinach [( 2Fe-2S]) and Clostridium pasteurianum (2[4Fe-4S]) ferredoxins in aqueous Triton X-100/urea containing excess benzenethiol yield quantitatively [FenSn(SPh)4]2- with n = 2 and n = 4, respectively. The iron-sulfur cluster can be separated from the corresponding apoprotein by rapid passage of the extrusion mixture over a small anaerobic column of Whatman DE-52 anion-exchange cellulose. Essentially quantitative recovery of [FenSn (SPh)4]2- is achieved in the eluate. The apoprotein remaining on the column can be eluted with 0.5 M NaCl. Most of the residual Triton X-100 and benzenethiol can be removed by passage of the apoprotein eluate over a small column of Bio-Beads SM-2, a hydrophobic polystyrene adsorbent. Apoprotein recovery is comparable to that obtained by other chromatographic methods. At least with spinach ferredoxin, the apoprotein prepared in this fashion can be reconstituted. The procedures developed in this work are potentially most applicable to selective removal of [2Fe-2S] and [4Fe-4S] centers from a multicenter enzyme without irreversible denaturation.

Metal substitutions at the diiron sites of hemerythrin and myohemerythrin: contributions of divalent metals to stability of a four-helix bundle protein.

A general method is described for substitution of Mn(II) and Co(II) into the diiron sites of hemerythrin and myohemerythrin. Characterizations of these metal-substituted proteins show that their structures closely resemble those of the native proteins. In particular, the four-helix bundle structure appears to be maintained. The apomyohemerythrin retains most of the native helix content but is considerably less stable to denaturation than are the metal-containing proteins. The relative affinities of M(II) for apohemerythrin--namely, Co greater than Fe greater than Mn--parallel the stabilities of the M2myohemerythrins to denaturation by guanidinium chloride. These results indicate that for myohemerythrin (i) the majority of the helical structure found in the native protein does not require incorporation of M(II) and (ii) stabilization of the native structure relative to the fully unfolded structure appears to be due predominantly to M(II)-protein interactions, at least for M = Fe and Co. Incorporation of M(II) also generates unfolding cooperativity in myohemerythrin. This cooperativity can be attributed to interhelical interactions, which are prevented in the apoprotein by solvation of the seven metal ligand residues. The results are consistent with a minimal model for folding/unfolding of myohemerythrin and hemerythrin subunits consisting of the sequential equilibria, N in equilibrium with I in equilibrium with D, between native, intermediate, and fully unfolded states, respectively. The properties of apomyohemerythrin make it a candidate for the intermediate state, I.

Kinetics and equilibria of active site core extrusion from spinach ferredoxin in aqueous N,N-dimethylformamide/Triton X-100 solutions.

The nature and reactivity of each species participating in core extrusion of the [2Fe-2S] cluster of spinach ferredoxin by benzenethiol have been investigated in a novel aqueous medium containing 10-40% (v/v) N,N-dimethylformamide (DMF) plus 5-6% (v/v) of the nonionic detergent, Triton X-100. By use of visible absorption and circular dichroism spectroscopies, it is found that prior to addition of benzenethiol, modifications of the ferredoxin induced by DMF are reversible. Both the extent and rate of modification are dependent on DMF concentration and on ionic strength. At ferredoxin concentrations near 0.1 mM, complete modification by DMF is shown to be unnecessary for quantitative core extrusion provided that the benzenethiol concentration is greater than or equal to 50 mM and the DMF concentration is greater than or equal to 10% (v/v). When benzenethiol is added after DMF, core extrusion occurs in two phases. Our interpretation is that the rapid phase corresponds to core extrusion of that portion of the ferredoxin already modified by DMF and that the slower phase corresponds to modification of the remaining portion of the ferredoxin by DMF. When DMF solutions containing benzenethiol and Triton X-100 in various ratios are mixed with aqueous solutions of spinach ferredoxin, the rate of core extrusion appears to be determined both by the rate at which the ferredoxin is modified and by the Triton/benzenethiol ratio. Under all conditions examined we observe significantly faster rates of core extrusion from spinach ferredoxin in aqueous Triton/DMF than in 80/20 (v/v) hexamethylphosphoramide/water, a previously used core extrusion medium. Our results suggest a catalytic role for the micellar phase.

Two distinct subunits of hemerythrin from the brachiopod Lingula reevii: an apparent requirement for cooperativity in O2 binding.

Reported are results on the subunit composition of octameric hemerythrin (Hr) from the brachiopod Lingula reevii. Unlike most other Hrs, L. reevii Hr shows cooperativity in O2 binding. Purified L. reevii Hr was found to consist of two different subunits in approximately equimolar proportions. These two subunits differ in molecular weight by approximately 1000. Amino acid sequence data for the first 24 residues of the two subunits, labeled alpha and beta, show 70% identity with each other. Comparisons to amino acid sequences of other Hrs show approximately 50% identity in the first 24 residues and that both the alpha and beta subunits of L. reevii Hr have one residue deleted at their amino termini. Very recently, one other Hr, that from the brachiopod Lingula unguis, was also shown to contain equimolar proportions of two different subunits [Satake, K., Yugi, M., Kamo, M., Kihara, H., & Tsugita, A. (1990) Protein Seq. Data Anal. 3, 1-5], and this Hr also shows cooperativity in O2 binding. An alpha 4 beta 4 octamer is, therefore, proposed to be a common feature of those Hrs that show such cooperativity. Likely arrangements of alpha and beta subunits within an alpha 4 beta 4 octamer having the same configuration of subunits as that in other octameric Hrs are proposed. The most probable arrangements can be readily derived from physically reasonable restrictions on the types of intersubunit interactions and on transmission of allosteric effects.