Influence of patient and donor cytokine genotypes on renal allograft rejection: evidence from a single centre study.

Cytokines are key immune mediators and it has been suggested that cytokine gene polymorphisms affecting expression influence rejection or tolerance. This study sought to examine this hypothesis with the aim of identifying predictive genotype markers for rejection. The study group consisted of 120 consecutive first cadaveric recipient-donor pairs transplanted at a single centre, between 1994 and 1997. PCR utilising sequence-specific primers (SSP) methodology was optimised for genotyping recipient and donor DNA for the following polymorphisms: tumour necrosis factor (TNF) -alpha (-308, G/A), interleukin (IL)-10 (-1082, G/A), IL-4 (-590, C/T), transforming growth factor (TGF) -beta1 (+915, G/C). Recipient-donor pairs were divided into rejectors (n = 28) and non-rejectors (n = 92). Each group was further stratified according to number of rejection episodes and HLA-DR mismatching. Recipient-donor pairs both lacking the IL-4*T allele (recipient low producer/donor low producer) were significantly increased in the rejector group (P = 0.02). Also, the combination of recipient IL-10*A negative/donor IL-10*A positive (recipient high producer/donor low producer), was significantly decreased in multiple rejectors (P = 0.04). No significant associations were detected between TNF-alpha and TGF-beta1, and rejection. This study suggests that the combination of recipient-donor IL-4 and IL-10 genotypes may be important in renal transplantation outcome. The results appear to corroborate the protective role of both of these cytokines, possibly due to their ability to suppress inflammation. However, due to conflicting results from this and other studies, a multi-centre collaborative study may be required to determine whether cytokine genotypes are significant, independent predictors of renal allograft rejection.

Simultaneous binding of PtdIns(4,5)P2 and clathrin by AP180 in the nucleation of clathrin lattices on membranes.

Adaptor protein 180 (AP180) and its homolog, clathrin assembly lymphoid myeloid leukemia protein (CALM), are closely related proteins that play important roles in clathrin-mediated endocytosis. Here, we present the structure of the NH2-terminal domain of CALM bound to phosphatidylinositol-4,5- bisphosphate [PtdIns(4,5)P2] via a lysine-rich motif. This motif is found in other proteins predicted to have domains of similar structure (for example, Huntingtin interacting protein 1). The structure is in part similar to the epsin NH2-terminal (ENTH) domain, but epsin lacks the PtdIns(4,5)P2-binding site. Because AP180 could bind to PtdIns(4,5)P2 and clathrin simultaneously, it may serve to tether clathrin to the membrane. This was shown by using purified components and a budding assay on preformed lipid monolayers. In the presence of AP180, clathrin lattices formed on the monolayer. When AP2 was also present, coated pits were formed.

Protection of radical intermediates at the active site of adenosylcobalamin-dependent methylmalonyl-CoA mutase.

Adenosylcobalamin-dependent methylmalonyl-CoA mutase catalyzes the interconversion of methylmalonyl-CoA and succinyl-CoA via radical intermediates generated by substrate-induced homolysis of the coenzyme carbon-cobalt bond. From the structure of methylmalonyl-CoA mutase it is evident that the deeply buried active site is completely shielded from solvent with only a few polar contacts made between the protein and the substrate. Site-directed mutants of amino acid His244, a residue close to the inferred site of radical chemistry, were engineered to investigate its role in catalysis. Two mutants, His244Ala and His244Gln, were characterized using kinetic and spectroscopic techniques. These results confirmed that His244 is not an essential residue. However, compared with that of the wild type, k(cat) was lowered by 10(2)- and 10(3)-fold for the His244Gln and His244Ala mutants, respectively, while the K(m) for succinyl-CoA was essentially unchanged in both cases. The primary kinetic tritium isotope effect (k(H)/k(T)) for the His244Gln mutant was 1.5 +/- 0.3, and tritium partitioning was now found to be dependent on the substrate used to initiate the reaction, indicating that the rearrangement of the substrate radical to the product radical was extremely slow. The His244Ala mutant underwent inactivation under aerobic conditions at a rate between 1 and 10% of the initial rate of turnover. The crystal structure of the His244Ala mutant, determined at 2.6 A resolution, indicated that the mutant enzyme is unaltered except for a cavity in the active site which is occupied by an ordered water molecule. Molecular oxygen reaching this cavity may lead directly to inactivation. These results indicate that His244 assists directly in the unusual carbon skeleton rearrangement and that alterations in this residue substantially lower the protection of reactive radical intermediates during catalysis.

Dislocation and hypertrophy of the medial head of the clavicle: an unusual late complication of radical neck dissection.

We report three cases of a rare late complication of neck dissection: anterior dislocation and hypertrophy of the sternal head of the clavicle manifesting as a hard lump in the lower neck. We describe the mode of presentation, etiology, and methods of prevention.

Crystal structure of substrate complexes of methylmalonyl-CoA mutase.

X-ray crystal structures of methylmalonyl-CoA mutase in complexes with substrate methylmalonyl-CoA and inhibitors 2-carboxypropyl-CoA and 3-carboxypropyl-CoA (substrate and product analogues) show that the enzyme-substrate interactions change little during the course of the rearrangement reaction, in contrast to the large conformational change on substrate binding. The substrate complex shows a 5'-deoxyadenine molecule in the active site, bound weakly and not attached to the cobalt atom of coenzyme B12, rotated and shifted from its position in the substrate-free adenosylcobalamin complex. The position of Tyralpha89 close to the substrate explains the stereochemical selectivity of the enzyme for (2R)-methylmalonyl-CoA.

A structural explanation for the recognition of tyrosine-based endocytotic signals.

Many cell surface proteins are marked for endocytosis by a cytoplasmic sequence motif, tyrosine-X-X-(hydrophobic residue), that is recognized by the mu2 subunit of AP2 adaptors. Crystal structures of the internalization signal binding domain of mu2 complexed with the internalization signal peptides of epidermal growth factor receptor and the trans-Golgi network protein TGN38 have been determined at 2.7 angstrom resolution. The signal peptides adopted an extended conformation rather than the expected tight turn. Specificity was conferred by hydrophobic pockets that bind the tyrosine and leucine in the peptide. In the crystal, the protein forms dimers that could increase the strength and specificity of binding to dimeric receptors.

Stabilization of radical intermediates by an active-site tyrosine residue in methylmalonyl-CoA mutase.

The adenosylcobalamin-dependent methylmalonyl-CoA mutase catalyzes the reversible rearrangement of methylmalonyl-CoA into succinyl-CoA by a free-radical mechanism. The recently solved X-ray crystal structure of methylmalonyl-CoA mutase from Propionibacterium shermanii has shown that tyrosine 89 is an active-site residue involved in substrate binding. The role of tyrosine 89, a conserved residue among methylmalonyl-CoA mutases, has been investigated by using site-directed mutagenesis to replace this residue with phenylalanine. The crystal structure of the Tyr89Phe mutant was determined to 2.2 A resolution and was found to be essentially superimposable on that of wild-type. Mutant and wild-type enzyme have very similar KM values, but kcat for the Tyr89Phe mutant is 580-fold lower than for wild-type. The rate of release of tritium from 5'-[3H]adenosylcobalamin during the enzymatic reaction and its rate of appearance in substrate and product were measured. The tritium released was found to partition unequally between methylmalonyl-CoA and succinyl-CoA, in a ratio of 40:60 when the reaction was initiated by addition of methylmalonyl-CoA and in a ratio of 10:90 when the reaction was initiated by addition of succinyl-CoA. The overall release of tritium was four times faster when succinyl-CoA was used as substrate. The tritium isotope effect on the enzyme catalyzed hydrogen transfer, measured with methylmalonyl-CoA as a substrate, was kH/kT = 30, which is within the expected range for a full primary kinetic tritium isotope effect. The different partitioning of tritium, dependent upon which substrate was used, and the normal value for the kinetic tritium isotope effect contrast markedly with the behavior of wild-type mutase. It appears that the loss of a single interaction involving the hydroxyl group of tyrosine 89 both affects the stability of radical intermediates and decreases the rate of interconversion of the substrate- and product-derived radicals.

Crystal structure of the spliceosomal U2B"-U2A' protein complex bound to a fragment of U2 small nuclear RNA.

We have determined the crystal structure at 2.4 A resolution of a ternary complex between the spliceosomal U2B"/U2A' protein complex and hairpin-loop IV of U2 small nuclear RNA. Unlike its close homologue the U1A protein, U2B" binds to its cognate RNA only in the presence of U2A', which contains leucine-rich repeats in its sequence. The concave surface of a parallel beta-sheet within the leucine-rich-repeat region of U2A' interacts with the ribonucleoprotein domain of U2B" on the surface opposite its RNA-binding surface. The basic carboxy-terminal region of U2A' interacts with the RNA stem. The crystal structure reveals how protein-protein interaction regulates RNA-binding specificity, and how replacing only a few key residues allows the U2B" and U1A proteins to discriminate between their cognate RNA hairpins by forming alternative networks of interactions.

Conformational changes on substrate binding to methylmalonyl CoA mutase and new insights into the free radical mechanism.

BACKGROUND: Methylmalonyl CoA mutase catalyses the interconversion of succinyl CoA and methylmalonyl CoA via a free radical mechanism. The enzyme belongs to a family of enzymes that catalyse intramolecular rearrangement reactions in which a group and a hydrogen atom on adjacent carbons are exchanged. These enzymes use the cofactor adenosylcobalamin (coenzyme B12) which breaks to form an adenosyl radical, thus initiating the reaction. Determination of the structure of substrate-free methylmalonyl CoA mutase was initiated to provide further insight into the mechanism of radical formation. RESULTS: We report here two structures of methylmalonyl CoA mutase from Propionibacterium shermanii. The first structure is of the enzyme in a nonproductive complex with CoA at 2.5 A resolution. This structure serves as a model for the substrate-free conformation of the enzyme, as it is very similar to the second much poorer 2.7 A resolution structure derived from a truly substrate-free crystal. The true substrate-free structure also shows the adenosyl group bound to the cobalt atom. Comparison of this structure with that of the previously reported complex of the enzyme with a substrate analogue shows that major conformational changes occur upon substrate binding. The substrate-binding site of the enzyme is located within a (beta alpha)8 TIM-barrel domain. In the absence of substrate, this TIM-barrel domain is split apart and the active site is accessible to solvent. When substrate binds, the barrel closes up with the substrate along its axis and the active site becomes completely buried. CONCLUSIONS: The closure of the active-site cavity upon substrate binding displaces the adenosyl group of the cofactor from the central cobalt atom into the active-site cavity. This triggers the formation of the free radical that initiates the rearrangement reaction. The TIM-barrel domain is substantially different from all others yet reported: in its unliganded form it is broken open, exposing the small hydrophilic sidechains which fill the centre. The typical barrel structure is only formed when substrate is bound.

Salvage treatment for inoperable neck nodes in head and neck cancer using combined iridium-192 brachytherapy and surgical reconstruction.

The results of debulking surgery and re-irradiation with radioactive implants (brachytherapy) are reported for 39 patients with inoperable metastatic neck nodes from primary head and neck cancers. For 13 patients conventional salvage by partial debulking surgery and brachytherapy proved effective, with 68 per cent control at 1 year, but six patients suffered severe radiation fibrosis, necrosis and contractures. Some 26 patients were treated by combined tumour debulking, skin resurfacing and brachytherapy implant. Initial tumour control and freedom from serious toxicity was achieved in 24 patients. Local control was achieved in 63 per cent of patients at 1 year, with a serious morbidity rate of 12 per cent.

How coenzyme B12 radicals are generated: the crystal structure of methylmalonyl-coenzyme A mutase at 2 A resolution.

BACKGROUND: The enzyme methylmalonyl-coenzyme A (CoA) mutase, an alphabeta heterodimer of 150 kDa, is a member of a class of enzymes that uses coenzyme B12 (adenosylcobalamin) as a cofactor. The enzyme induces the formation of an adenosyl radical from the cofactor. This radical then initiates a free-radical rearrangement of its substrate, succinyl-CoA, to methylmalonyl-CoA. RESULTS: Reported here is the crystal structure at 2 A resolution of methylmalonyl-CoA mutase from Propionibacterium shermanii in complex with coenzyme B12 and with the partial substrate desulpho-CoA (lacking the succinyl group and the sulphur atom of the substrate). The coenzyme is bound by a domain which shares a similar fold to those of flavodoxin and the B12-binding domain of methylcobalamin-dependent methionine synthase. The cobalt atom is coordinated, via a long bond, to a histidine from the protein. The partial substrate is bound along the axis of a (beta/alpha)8 TIM barrel domain. CONCLUSIONS: The histidine-cobalt distance is very long (2.5 A compared with 1.95-2.2 A in free cobalamins), suggesting that the enzyme positions the histidine in order to weaken the metal-carbon bond of the cofactor and favour the formation of the initial radical species. The active site is deeply buried, and the only access to it is through a narrow tunnel along the axis of the TIM barrel domain.

Site-directed mutagenesis identifies catalytic residues in the active site of Escherichia coli phosphofructokinase.

Six active site mutants of Escherichia coli phosphofructokinase have been constructed and characterized using steady-state kinetics. All but one of the mutants (ES222) have significantly lower maximal activity, implicating these residues in the catalytic process. Replacement of Asp127, the key catalytic residue in the forward reaction with Glu, results in an enzyme with wild-type cooperative and allosteric behavior but severely decreased Fru6P binding. Replacement of the same residue with Tyr abolishes cooperativity while retaining sensitivity to allosteric inhibition and activation. Thus, this mutant has uncoupled homotropic from heterotropic allostery. Mutation of Asp103 to Ala results in an enzyme which retains wild-type Fru6P-binding characteristics with reduced activity. GDP, which allosterically activates the wild-type enzyme, acts as a mixed inhibitor for this mutant. Mutation of Thr125 to Ala and Asp129 to Ser produces mutants with impaired Fru6P binding and decreased cooperativity. In the presence of the activator GDP, both these mutants display apparent negative cooperativity. In addition, ATP binding is now allosterically altered by GDP. These results extend the number of active site residues known to participate in the catalytic process and help to define the mechanisms behind catalysis and homotropic and heterotropic allostery.

Lack of correlation between IgG T-lymphocyte flow cytometric crossmatches with primary renal allograft outcome.

The flow cytometric crossmatch (FCXM) has been reported to be more sensitive and capable of detecting very low levels of antibodies than the normally used complement dependent cytotoxicity test. We studied both the two colour IgG T cell FCXM and CDC-XM in 146 renal allograft recipients, 111 primary and 35 regrafts, of which 26% (29/111) of 1st and 20% (7/35) of regrafts had a positive FCXM. There was no overall correlation between the FCXM results and early graft outcome in primary renal allografts. The FCXM did not appear to have any advantage over the CDC-XM in predicting graft outcome in unsensitized first grafts. In the small number of regrafts studied, a positive FCXM was associated with a higher degree of graft failure. FCXM can exhibit false negative results if sera are used solely neat although these prozone phenomena do not influence subsequent graft outcome.

Steady-state fluorescence of Escherichia coli phosphofructokinase reveals a regulatory role for ATP.

We have investigated the effects of ligands and effectors on the intrinsic fluorescence of Escherichia coli phosphofructokinase (PFK). We have found that the substrate fructose 6-phosphate (Fru6P) or the allosteric activator ADP can quench the fluorescence up to 35%. The response is hyperbolic with Ks[Fru6P] of 20 microM and Ks[ADP] of 13 microM. The allosteric inhibitor phosphoenolpyruvate (PEP) converts the hyperbolic response with respect to Fru6P to a sigmoidal response. AMP-PNP, a nonhydrolyzable analogue of ATP, also inhibits the Fru6P fluorescence response. PFK mutant KA213, which is insensitive to effectors, has a decreased fluorescence response with respect to ADP, and PEP does not convert the Fru6P response to sigmoidicity. However, its fluorescence response with respect to Fru6P is decreased by ATP or AMP-PNP. Taken together, these results suggest that, in the absence of effectors or ligands, E. coli PFK exists in a state with high affinity for Fru6P ("R" state). This state can be altered to a low affinity ("T" state) by PEP binding to the allosteric site or by ATP binding to the enzyme.

Designing an allosterically locked phosphofructokinase.

Six site-directed mutants of Escherichia coli phosphofructokinase (PFK) were made in an attempt to produce an enzyme "locked" in the inactive or "T"-state. The kinetic properties of the mutants were examined as a function of the substrates fructose 6-phosphate (Fru6P) and ATP, the positive effector GDP, and the negative effector phosphoenolpyruvate (PEP). All mutants exhibited lower activity than wild-type PFK. Three mutants (RS63, LV153, and VT246) had apparent dissociation constants for substrates and effectors similar to those of wild type. One mutant, HN160, had a 10-fold reduced affinity for Fru6P and reduced apparent affinity for the effectors. Two mutants, SN159 and T(GS)156, exhibited hyperbolic kinetics consistent with a "locked" T-state protein. Surprisingly, T(GS)156 showed hyperbolic activation in response to the physiological inhibitor PEP. The mutant PFK properties are discussed in terms of the PFK structure. These results suggest that the kinetic properties of PFK are sensitive to interactions in the homotropic interface; residues 156-160 in particular are critical in mediating the interactions between effector and active sites and in the T to R quaternary transition.

Villous adenomas of the duodenum and an unusual variant.

We report two cases of villous adenoma of the duodenum, one arising from the main papilla and the other from the accessory papilla. Both were managed by local resection. In one case endoscopic biopsies and intraoperative frozen sections were negative for carcinoma but histology of the locally resected specimen revealed a focus of invasive adenocarcinoma. Villous adenomas of the duodenum have a high risk of malignant change and foci of carcinoma can be missed on endoscopic biopsy. The literature is reviewed and the clinical, diagnostic, pathological and therapeutic aspects of villous adenomas of the duodenum are discussed.

Sperm morphological analysis: comparison of two methods in human semen.

Sperm morphological analysis was performed by 2 methods: light microscopy on Papanicolaou stained material and scanning electron microscopy. Both methods were found to have acceptably low levels of observer error and a positive correlation existed between them. Sperm morphology was analysed by both methods in 2 groups: (1) 104 proven "fertile" men, husbands of women who had just given birth and whose paternity had been proven by HLA studies with greater than 95.5% certainty; (2) 53 infertile men from primary infertile marriages in whom no female cause could be found and excluding those with azoospermia. There was no statistically significant difference in medians between the 2 groups when sperm morphology was analysed by light microscopy, but analysis by scanning electron microscopy did show a significant difference.

Active-site mutants altering the cooperativity of E. coli phosphofructokinase.

Crystal structures of the high- and low-activity states of the allosteric enzyme phosphofructokinase implicate three arginines in substrate binding, catalysis and cooperativity. Arginines 162 and 243 reach into the active site from an adjacent subunit and interact with the cooperative substrate fructose 6-phosphate. Mutation of these arginines to serine results in mutant enzymes with reduced substrate binding and lowered cooperativity, but with little change in their catalytic ability (kcat). Arg 72 bridges the two substrates fructose 6-phosphate and ATP, and interacts with the 1-phosphate of the product fructose 1,6-biphosphate. Mutation of this residue to serine reduces the catalytic activity, cooperativity and binding of fructose 6-phosphate and fructose 1,6-bisphosphate. In the reverse reaction, the kinetics of wild-type and the Ser 72 mutant with respect to fructose 1,6-bisphosphate are hyperbolic, whereas those of the Ser 162 and Ser 243 mutants are sigmoidal. These results show that each of the three arginines contributes to cooperativity and to the transmission of allosteric signals between the four subunit of the enzyme.