Intracranial hematolymphoid malignancies: A case series with molecular characterization.

OBJECTIVE: Central nervous system (CNS) manifestations of hematologic malignancies are uncommon and often have a poor prognosis. As hematologic neoplasms are typically chemotherapy- and radiotherapy-sensitive, surgical resection is usually not indicated; thus, opportunities for in-depth characterization of CNS hematologic tumors are limited. Here, we report four cases of rare intracranial hematologic tumors requiring surgical intervention, allowing for histopathologic and genomic characterization. METHODS: The clinical course, genetic perturbations, and histopathological features are described for a case of 1) primary marginal zone B-cell lymphoma of the dura as well as cases of brain metastases of 2) cutaneous T-cell lymphoma, 3) acute myeloid leukemia/myeloid sarcoma, and 4) multiple myeloma. Targeted DNA sequencing, fluorescence in situ hybridization, cytogenetic analysis, flow cytometry and immunohistochemical staining were used to assess the lesions. RESULT: Molecular and histopathological characterizations of four unusual presentations of hematolymphoid diseases involving the CNS are presented. Genetic abnormalities were identified in each lesion, including chromosomal aberrations and single nucleotide variants resulting in missense or nonsense mutations in oncogenes. CONCLUSIONS: Our case series provides insight into unique pathological phenotypes of hematologic neoplasms with atypical CNS involvement. We offer targets for future studies by identifying potentially pathogenic genetic variants in these lesions, as the full implications of the novel molecular abnormalities described remain unclear.

Evaluation of the effect of bacterial efflux pumps on the antibacterial activity of the novel fluoroquinolone besifloxacin.

This study examined the susceptibility of a variety of wild-type strains and efflux pump mutants to besifloxacin and the comparator agents sparfloxacin, ciprofloxacin, norfloxacin, moxifloxacin, tetracycline, and ethidium bromide. Organisms tested included Staphylococcus aureus (mepA or norA), Streptococcus pneumoniae (pmrA, patB), Escherichia coli (acrAB::Tn903, tolC::Tn10), Haemophilus influenzae (acrAB) and Pseudomonas aeruginosa (mepAB-oprM, oprM::ΩHg(r) rpsL). The minimal inhibitory concentrations (MIC) of besifloxacin and comparators were also measured in the presence of the efflux pump inhibitors reserpine, carbonyl cyanide mchlorophenyl- hydrazone, or sodium orthovanadate. Overall, very few meaningful changes (>2-fold) in besifloxacin MIC values resulted from the presence of efflux pump mutations or efflux pump inhibitors. In summary, the novel fluoroquinolone besifloxacin is no exception to the observation that newer fluoroquinolones are generally less affected by efflux pump-mediated resistance than older fluoroquinolones.

Staphylococcus aureus mutants isolated via exposure to nonfluorinated quinolones: detection of known and unique mutations.

The in vitro development of resistance to the new nonfluorinated quinolones (NFQs; PGE 9262932, PGE 4175997, and PGE 9509924) was investigated in Staphylococcus aureus. At concentrations two times the MIC, step 1 mutants were isolated more frequently with ciprofloxacin and trovafloxacin (9.1 x 10(-8) and 5.7 x 10(-9), respectively) than with the NFQs, gatifloxacin, or clinafloxacin (<5.7 x 10(-10)). Step 2 and step 3 mutants were selected via exposure of a step 1 mutant (selected with trovafloxacin) to four times the MICs of trovafloxacin and PGE 9262932. The step 1 mutant contained the known Ser80-Phe mutation in GrlA, and the step 2 and step 3 mutants contained the known Ser80-Phe and Ser84-Leu mutations in GrlA and GyrA, respectively. Compared to ciprofloxacin, the NFQs were 8-fold more potent against the parent and 16- to 128-fold more potent against the step 3 mutants. Mutants with high-level NFQ resistance (MIC, 32 microg/ml) were isolated by the spiral plater-based serial passage technique. DNA sequence analysis of three such mutants revealed the following mutations: (i) Ser84-Leu in GyrA and Glu84-Lys and His103-Tyr in GrlA; (ii) Ser-84Leu in GyrA, Ser52-Arg in GrlA, and Glu472-Val in GrlB; and (iii) Ser84-Leu in GyrA, Glu477-Val in GyrB, and Glu84-Lys and His103-Tyr in GrlA. Addition of the efflux pump inhibitor reserpine (10 microg/ml) resulted in 4- to 16-fold increases in the potencies of the NFQs against these mutants, whereas it resulted in 2-fold increases in the potencies of the NFQs against the parent.

The identification and characterization of hydrazinyl urea-based antibacterial agents through combinatorial chemistry.

An effort to identify novel inhibitors of peptidoglycan synthesis with antibacterial activity resulted in the discovery of a series of biaryl urea-based antibacterial agents through isolation of a by-product from a mixture-based combinatorial library of semi-carbazones and subsequent parallel synthesis efforts. The compounds were shown to possess broad spectrum antibacterial activity against gram-positive drug resistant pathogens, and showed apparent specificity for disruption of the bacterial cell wall biosynthesis pathway.

Effect of angiotensin converting enzyme inhibition on the Doppler waveform in dogs with renal artery stenosis.

Our objective was to investigate whether the angiotensin converting enzyme inhibitor enalaprilat improves detection of hemodynamically significant renal artery stenoses in dogs. Renal artery stenoses of 50 to 99% were surgically created unilaterally in five dogs. Doppler ultrasonographic evaluation was performed at baseline (no stenosis), after creation of the stenosis, and after the administration of enalaprilat. The resistive index increased in the nonstenotic kidney (P < 0.01) but not in the stenotic kidney after administration of enalaprilat. The difference in resistive indices between nonstenotic and stenotic kidneys increased significantly (P < 0.05) after administration of enalaprilat. Measurement of the resistive index after administration of an angiotensin converting enzyme inhibitor in humans may improve the performance of Doppler ultrasonography in detecting hemodynamically significant renal artery stenoses.

Application of nearest-neighbor and cluster analyses in pharmaceutical lead discovery.

High throughput screening (HTS) programs based on diverse collections of compounds can rapidly identify leads for potential drug candidates. In cases where the compound collection is truly diverse, one may only identify a few compounds of interest. However, where a large number of hits are identified, it becomes necessary to examine the structures to determine the true number of compound classes involved so that follow-up studies may be conducted as efficiently as possible. In this case, cluster analysis is applied to determine the structural relationship among HTS hits. To efficiently expand around the region of the hit (or a class of hits) in chemical space, we have applied nearest neighbors analysis to select additional compounds from collections of a large number of commercial vendors, achieving an average hit rate in excess of 15%. Applying these techniques in a number of different cases, we obtained results that are useful for subsequent investigations of hits from HTS and other relevant molecular structures from the literature.

Molecular recognition in a post-translational modification of exceptional specificity. Mutants of the biotinylated domain of acetyl-CoA carboxylase defective in recognition by biotin protein ligase.

We have used localized mutagenesis of the biotin domain of the Escherichia coli biotin carboxyl carrier protein coupled with a genetic selection to identify regions of the domain having a role in interactions with the modifying enzyme, biotin protein ligase. We purified several singly substituted mutant biotin domains that showed reduced biotinylation in vivo and characterized these proteins in vitro. This approach has allowed us to distinguish putative biotin protein ligase interaction mutations from structurally defective proteins. Two mutant proteins with glutamate to lysine substitutions (at residues 119 or 147) behaved as authentic ligase interaction mutants. The E119K protein was virtually inactive as a substrate for biotin protein ligase, whereas the E147K protein could be biotinylated, albeit poorly. Neither substitution affected the overall structure of the domain, assayed by disulfide dimer formation and trypsin resistance. Substitutions of the highly conserved glycine residues at positions 133 and 143 or at a key hydrophobic core residue, Val-146, gave structurally unstable proteins.

Luminal diameter and vasa vasorum response to stent dilation in the rabbit aorta.

RATIONALE AND OBJECTIVES: We examined the changes in luminal diameter and vasa vasorum after stent dilation of the aorta of nonatherosclerotic rabbits. METHODS: Balloon-expandable stents were placed in the aortas of rabbits at the L3 level and were expanded. After 8 weeks, the rabbits were euthanized, perfused with barium sulfate particles, frozen, sectioned, and X-rayed. RESULTS: The luminal intrastent diameter (2.3 +/- 0.23 mm) was significantly different (p < .05) from the expanded stent diameter but was not different from the luminal diameter above (2.3 +/- 0.53 mm) and below (1.9 +/- 0.18 mm) the stent. The number of vasa vasorum at the stent (46 +/- 7.2) was significantly increased compared with above (21 +/- 4.8, p < .001) and below (14.4 +/- 4.6, p < .001) the stent. CONCLUSION: After 8 weeks, luminal diameter was uniform. The increased vasa vasorum in the stented section suggested an increase in blood flow to the aortic wall, which may be important to the remodeling process.

Purification and properties of the lipoate protein ligase of Escherichia coli.

Lipoate is an essential component of the 2-oxoacid dehydrogenase complexes and the glycine-cleavage system of Escherichia coli. It is attached to specific lysine residues in the lipoyl domains of the E2p (lipoate acetyltransferase) subunit of the pyruvate dehydrogenase complex by a Mg(2+)- and ATP-dependent lipoate protein ligase (LPL). LPL was purified from wild-type E. coli, where its abundance is extremely low (< 10 molecules per cell) and from a genetically amplified source. The purified enzyme is a monomeric protein (M(r) 38,000) which forms irregular clusters of needle-like crystals. It is stable at -20 degrees C, but slowly oxidizes to an inactive form containing at least one intramolecular disulphide bond at 4 degrees C. The inactive form could be re-activated by reducing agents or by an as-yet unidentified component (reactivation factor) which is resolved from LPL at the final stage of purification. The pI is 5.80, and the Km values for ATP, Mg2+ and DL-lipoate were determined. Selenolipoate and 6-thio-octanoate were alternative but poorer substrates. Lipoylation was reversibly inhibited by the 6- and 8-seleno-octanoates and 8-thio-octanoate, which reacted with the six cysteine thiol groups of LPL. LPL was inactivated by Cu2+ ions in a process that involved the formation of inter- and intra-molecular disulphide bonds. Studies with lplA mutants lacking LPL activity indicated that E. coli possesses another distinct lipoylation system, although no such activity could be detected in vitro.

Lipoic acid metabolism in Escherichia coli: the lplA and lipB genes define redundant pathways for ligation of lipoyl groups to apoprotein.

Lipoic acid is a covalently bound disulfide-containing cofactor required for function of the pyruvate dehydrogenase, alpha-ketoglutarate dehydrogenase, and glycine cleavage enzyme complexes of Escherichia coli. Recently we described the isolation of the lplA locus, the first gene known to encode a lipoyl-protein ligase for the attachment of lipoyl groups to lipoate-dependent apoenzymes (T. W. Morris, K. E. Reed, and J. E. Cronan, Jr., J. Biol. Chem. 269:16091-16100, 1994). Here, we report an unexpected redundancy between the functions of lplA and lipB, a gene previously identified as a putative lipoate biosynthetic locus. First, analysis of lplA null mutants revealed the existence of a second lipoyl ligase enzyme. We found that lplA null mutants displayed no growth defects unless combined with lipA (lipoate synthesis) or lipB mutations and that overexpression of wild-type LplA suppressed lipB null mutations. Assays of growth, transport, lipoyl-protein content, and apoprotein modification demonstrated that lplA encoded a ligase for the incorporation of exogenously supplied lipoate, whereas lipB was required for function of the second lipoyl ligase, which utilizes lipoyl groups generated via endogenous (lipA-mediated) biosynthesis. The lipB-dependent ligase was further shown to cause the accumulation of aberrantly modified octanoyl-proteins in lipoate-deficient cells. Lipoate uptake assays of strains that overproduced lipoate-accepting apoproteins also demonstrated coupling between transport and the subsequent ligation of lipoate to apoprotein by the LplA enzyme. Although mutations in two genes (fadD and fadL) involved in fatty acid failed to affect lipoate utilization, disruption of the smp gene severely decreased lipoate utilization. DNA sequencing of the previously identified slr1 selenolipoate resistance mutation (K. E. Reed, T. W. Morris, and J. E. Cronan, Jr., Proc. Natl. Acad. Sci. USA 91:3720-3724, 1994) showed this mutation (now called lplA1) to be a G76S substitution in the LplA ligase. When compared with the wild-type allele, the cloned lplA1 allele conferred a threefold increase in the ability to discriminate against the selenium-containing analog. These results support a two-pathway/two-ligase model of lipoate metabolism in E. coli.

A review of coronary arteriography- and contrast media-induced ventricular fibrillation.

Since the first animal coronary arteriogram in 1933 there have been many innovations in techniques and contrast media. From 1933 through the late 1950s the procedures used involved nonselective aortic injections and the use of acetylcholine to slow the heart. The first selective coronary arteriogram in animals was performed by West, Kobayashi & Guzman in 1958 (45) and in 1959 Guzman & West (7) observed ventricular fibrillation with some media but not others. In 1967 Judkins (14) described the catheter designs for right and left coronary catheterizations that we still use today. In the 1970s and 80s many authors observed the ionic monomeric contrast media reduced plasma calcium causing fibrillation and myocardial depression. Supplementation of ionic media with calcium was shown to moderate these adverse effects. Almen's vision of low osmolality contrast media and the creation of metrizamide (1) stimulated the rapid development of monomeric and dimeric nonionic contrast media. The ionic dimeric medium ioxaglate also provided low osmolality. Digital frame grabbers and computers lead to the development of digital subtraction angiography and new applications of arteriography, frequently using dilute media. Unexpectedly, during prolonged right coronary arteriography in animals, dilute nonionic media were found to produce increased fibrillation as compared to dilute ionic media. The addition of sodium to nonionic media significantly reduced the incidence of fibrillation. Animal studies with the nonionic medium iodixanol supplemented with sodium and calcium (Visipaque) have demonstrated minimal incidences of fibrillation and myocardial depression.

Cardiac hemodynamic effects of iodixanol, iopamidol, and ioxaglate following left coronary injections in anesthetized dogs.

RATIONALE AND OBJECTIVES: Iodixanol, a dimeric, nonionic X-ray contrast medium, has been formulated at 320 mg iodine per milliliter and supplemented with Na+, Ca2+, and Cl- to produce an osmolality that approximates that of plasma. We compared the effects of left main coronary artery injections of iodixanol, ioxaglate, and iopamidol on cardiac mechanical function in dogs. METHODS: Six mixed-breed dogs were anesthetized and prepared for recordings for electrocardiogram, aortic and left ventricular pressures, and the first derivative of left ventricular pressure, dP/dt. The test solutions and saline were injected into the left coronary artery in a randomized order. The series of four injections were repeated three times in each animal for a total of 12 injections per dog. RESULTS: Iodixanol caused significantly lower (p < .05) reduction in peak left ventricular pressure (-1.7 +/- 0.9% vs -0.7 +/- 2.0%), in diastolic aortic pressure (-1.3 +/- 1.1% vs -9 +/- 1.3%), and in left ventricular dP/dt (0.3 +/- 1.3% vs -13.2 +/- 2.4%) than did ioxaglate. Iodixanol also produced smaller cardiovascular effects than did iopamidol, but the differences were not statistically significant. Injections of both iopamidol and ioxaglate caused significant decreases from baseline parameter values; however, the changes with iodixanol were not significant. CONCLUSION: The isotonic formulation of iodixanol caused smaller cardiovascular hemodynamic effects than did iopamidol and ioxaglate and may offer increased safety in patients with severe cardiac disease.

Angiography of the temporomandibular joint. Description of an experimental technique with initial results.

The vascular supply to the temporomandibular joint is not completely understood. To form a base for advancement in this area we developed a method for experimental angiography of the temporomandibular joint that was applied to fresh temporomandibular joint autopsy specimens. Via the external carotid artery the vessels were infused with a mixture of barium and an acrylic resin. The specimens were sectioned and contact radiographs were obtained. These showed the vascularity of the joint and the surrounding structures with great detail. Most of the vascular supply appears to come from the lateral and medial aspects of the condyle head and from the anterior and posterior disk attachments. The method was applied to both normal and abnormal joints and the results suggest that this method could be used to gather further understanding of the vascularity of the temporomandibular joint relative to disease.

Noninvasive measurement of renal hemodynamic functions using gadolinium enhanced magnetic resonance imaging.

A technique for the assessment of single kidney hemodynamic functions utilizing a novel MR pulse sequence in conjunction with MR contrast material administration is described. Renal extraction fraction (EF) is derived by measuring the concentration of the incoming contrast agent in the renal artery and the outgoing concentration in the renal vein. The glomerular filtration rate (GFR) can then be determined by the product of EF and renal plasma flow. A modified inversion recovery MR pulse sequence is used to measure the T1 of moving blood. This pulse sequence uses a spatially nonselective inversion pulse. A series of small flip angle detection pulses are then used to monitor the recovery of longitudinal spin magnetization in an image plane intersecting the renal vessels. The recovery rate is measured in each vessel and the T1 of blood determined. These T1 measurements are then used to determine the ratio of contrast concentration in the renal arteries and veins. Blood flow measurements can be obtained simultaneously with T1 measurements by inserting flow-encoding magnetic field gradients into the pulse sequence. Preliminary results in human volunteers suggest the feasibility of noninvasively determining hemodynamic functions with magnetic resonance.

Expression, biotinylation and purification of a biotin-domain peptide from the biotin carboxy carrier protein of Escherichia coli acetyl-CoA carboxylase.

A protein segment consisting of the C-terminal 87 residues of the biotin carboxy carrier protein from Escherichia coli acetyl-CoA carboxylase was overexpressed in E. coli. The expressed biotin-domain peptide can be fully biotinylated by coexpression with a plasmid that overproduces E. coli biotin ligase. The extent of biotinylation was limited in vivo, but could be taken to completion in cell lysates on addition of ATP and biotin. We used the coexpression of biotin ligase and acceptor protein to label the biotin-domain peptide in vitro with [3H]biotin, which greatly facilitated development of a purification procedure. The apo (unbiotinylated) form of the protein was prepared by induction of biotin-domain expression in a strain lacking the biotin-ligase-overproduction plasmid. The apo domain could be separated from the biotinylated protein by ion-exchange chromatography or non-denaturing PAGE, and was converted into the biotinylated form of the peptide on addition of purified biotin ligase. The identify of the purified biotin-domain peptide was confirmed by N-terminal sequence analysis, amino acid analysis and m.s. The domain was readily produced and purified in sufficient quantities for n.m.r. structural analysis.

Identification of the gene encoding lipoate-protein ligase A of Escherichia coli. Molecular cloning and characterization of the lplA gene and gene product.

R(+)-Lipoic acid is a cofactor required for function of the alpha-keto acid dehydrogenase and glycine cleavage enzyme complexes. The naturally occurring form of lipoate is attached by amide linkage to the epsilon-amino group of a specific lysine residue within conserved lipoate-accepting protein domains. Lipoate-protein ligase(s) catalyze the formation of this amide bond between lipoyl groups and specific apoproteins. We report the isolation of the lplA gene which encodes an Escherichia coli lipoate-protein ligase. Strains with lplA null mutations transport lipoic acid normally but have severe defects in the incorporation and utilization of exogenously supplied lipoic acid and lipoic acid analogs. These strains are also highly resistant to selenolipoate (a growth-inhibiting lipoate analog) and contain no detectable lipoate-protein ligase activity in cell extracts. The lplA gene has been cloned, sequenced, and physically mapped to min 99.6 (4657 kilobases) of the E. coli chromosome. Upon overexpression, the 38-kDa lplA gene product was purified to homogeneity and shown to have a mass, N-terminal sequence and amino acid composition consistent with the deduced 337 residue primary sequence. Enzyme assays show that purified LplA catalyzes the ATP-dependent attachment of [35S]lipoic acid to apoprotein, thus confirming that lplA encodes lipoate-protein ligase A. Analysis of lplA null mutants also indicates the existence of a second (lplA-independent) lipoyl-ligase enzyme in E. coli. This is the first identification of a lipoate ligase gene and the first analysis of a purified lipoate ligase enzyme.

Mutants of Escherichia coli K-12 that are resistant to a selenium analog of lipoic acid identify unknown genes in lipoate metabolism.

Lipoic acid is a disulfide-containing cofactor required for the reactions catalyzed by alpha-ketoacid dehydrogenase enzyme complexes. We report the chemical synthesis and biological properties of lipoic acid analogs in which one or both sulfur atoms were replaced by selenium. Replacement of either the C-6 or the C-8 sulfur atom with selenium results in lipoic acid derivatives with apparently unaltered biological properties. However, simultaneous replacement of both sulfur atoms gave an analog (selenolipoic acid) that inhibited growth of wild-type Escherichia coli when present in minimal glucose medium at 50 ng/ml. This growth inhibition was reversed by the addition of either excess lipoic acid or acetate plus succinate. Labeling experiments with [75Se]selenolipoic acid showed that this compound was efficiently incorporated into the alpha-ketoacid dehydrogenase complexes of growing cells. Spontaneously arising selenolipoic acid-resistant (slr) mutants were isolated. Two of these isolates resistant to high levels of selenolipoic acid were studied in detail. The slr-1 mutation, which was mapped to min 99.6 of the E. coli chromosome, increased the lipoate requirement of lipA strains by 4-fold and appeared to define a gene encoding a lipoate-protein ligase. The slr-7 mutation, which was mapped to min 15.25 of the chromosome, completely suppressed the lipoate requirement of lipA strains and defined a gene of unknown function in the synthesis of lipoic acid.