High reliability in digital planning of medial opening wedge high tibial osteotomy, using Miniaci's method.

PURPOSE: Pre-operative planning is essential in high tibial osteotomy (HTO). Miniaci's method employs Mikulicz's weight-bearing line and is advantageous because the point of mechanical loading is related to the known degenerative condition of the knee. Miniaci's geometrical method has been modified for an opening wedge and described for use with a digital picture archiving and communications system viewer. Reliability for this method was hypothesised to be equivalent to published reliability for landmark-based commercial software and independent of observer experience. METHODS: Twenty-four patients awaiting HTO had standardised long-leg radiographs. Mikulicz's weight-bearing line was projected through the lateral compartment of the knee at Fujisawa's point. The correction angle was generated at the hinge point subtending the current and proposed ankle centres. The opening wedge was plotted to measure an opening distance. Observations were recorded twice by three observers. Agreement was reported as intraclass correlation coefficients with 95 % confidence intervals. RESULTS: Intra-rater agreement was excellent for the correction angle (0.965-0.985) and opening distance (0.928-0.980). If no set hinge point was used, then the inter-rater reliability was 0.986 for the correction angle and 0.984 for the opening distance. There was no discernible pattern demonstrating improved reliability from the experienced observer. CONCLUSIONS: Reliability is comparable to commercially based landmark software and independent of observer experience. This makes such geometrical pre-operative planning accessible to surgeons who perform HTO with insufficient frequency to justify the investment in commercial software. LEVEL OF EVIDENCE: Diagnostic study, Level II.

Cholinergic sensitivity: normal variability as a function of stimulus background.

The sensitivity of the normally innervated iris sphincter to its neuro-transmitter, acetylchloline, and to relatd agents varies inversely with the preexisting physiological stimulus background, that is, the environmental light intnsity. This normal variability suggests the existence of a negative feedback mechahnism whereby sensitivity of the effector cell is modutlated by a product of neuronal activity.

Comparison of different busulfan analogues for depletion of hematopoietic stem cells and promotion of donor-type chimerism in murine bone marrow transplant recipients.

Busulfan (1,4-butanediol dimethanesulfonate, BU) is relatively unique among other standard chemotherapy compounds in its ability to deplete noncycling primitive stem cells in the host and consequently to allow for high levels of long-term, donor-type engraftment after bone marrow transplantation (BMT). Such a property explains why this drug can be used as an alternative to total body irradiation in preparative regimes for BMT. However, as with radiation, BU conditioning is still troubled by severe toxicities that limit its applications to suboptimal drug doses. These problems stress the need for other BMT-conditioning drugs that are better tolerated and more selectively targeted toward normal and malignant hematopoietic stem cells. We have therefore compared the effects of various novel dimethanesulfonate compounds (related to BU) in terms of their toxicity to different stem cell subsets in vivo and in vitro and their ability to provide for long-term donor bone marrow engraftment using the congenic glucose-6-phosphate isomerase type 1 marker. Introduction of a benzene or cyclohexane ring in some of these drugs affords rigidity to the molecule and restricts the spatial positioning of the alkylating groups. Among 25 different compounds thus far tested at single doses, PL63 [cis-1,2-(2-hydroxyethyl) cyclohexane dimethanesulfonate] proved to be the most effective in providing for hematopoietic engraftment. The transisomer of the same compound gave significantly less engraftment and was comparable with the effects of dimethylbusulfan and Hepsulfam. The engraftment data correlated well with the depletion of different bone marrow stem cell subsets in the host as measured using the cobblestone area forming cell assay. The extent of stem cell depletion could not be explained on the basis of the distance and orientation of the two alkylating groups. Pharmacokinetic data, however, indicate that there is a correlation between biological activity and plasma levels reached. The diverse cytotoxic effects shown by these novel analogues of BU have provided a basis for relating biological activity with pharmacokinetic properties rather than with structural properties such as distance and orientation of the two alkylating groups. The identification of highly active compounds such as PL63 offers an opportunity for further developing other closely related drugs for potential application in clinical BMT conditioning therapy.

Novel octaketide macrolides related to 6-deoxyerythronolide B provide evidence for iterative operation of the erythromycin polyketide synthase.

BACKGROUND: The macrolide antibiotic erythromycin A, like other complex aliphatic polyketides, is synthesised by a bacterial modular polyketide synthase (PKS). Such PKSs, in contrast to other fatty acid and polyketide synthases which work iteratively, contain a separate set or module of enzyme activities for each successive cycle of polyketide chain extension, and the number and type of modules together determine the structure of the polyketide product. Thus, the six extension modules of the erythromycin PKS (DEBS) together catalyse the production of the specific heptaketide 6-deoxyerythronolide B. RESULTS: A mutant strain of the erythromycin producer Saccharopolyspora erythraea, which accumulates the aglycone intermediate erythronolide B, was found unexpectedly to produce two novel octaketides, both 16-membered macrolides. These compounds were detectable in fermentation broths of wild-type S. erythraea, but not in a strain from which the DEBS genes had been specifically deleted. From their structures, both of these octaketides appear to be aberrant products of DEBS in which module 4 has 'stuttered', that is, has catalysed two successive cycles of chain extension. CONCLUSIONS: The isolation of novel DEBS-derived octaketides provides the first evidence that an extension module in a modular PKS has the potential to catalyse iterative rounds of chain elongation like other type I FAS and PKS systems. The factors governing the extent of such 'stuttering' remain to be determined.

HIN-200: a novel family of IFN-inducible nuclear proteins expressed in leukocytes.

In recent years a novel family of interferon (IFN)-inducible nuclear proteins has been identified in both mouse and humans and implicated in the control of myeloid differentiation. Recent evidence suggests that this role may be mediated by an effect on cell cycle regulation through interaction with known transcriptional regulators. Our laboratory first identified a nuclear protein designated IFI 16, which is strongly inducible with IFN-alpha and -gamma in myeloid cells. IFI 16 shares homology with at least six IFN-inducible mouse genes, designated the Gene 200 cluster, and also has close similarity with the human myeloid cell nuclear differentiation antigen, whose expression has also been associated with later stages of normal myelopoiesis and some myeloid malignancies. The structural hallmark of each protein in this family of hemopoietic IFN-inducible nuclear proteins is a 200-amino acid motif (hence HIN-200), present singly or as a tandem repeat. The restricted expression of these proteins, their ability to bind to nucleic acid, and their expression in response to a variety of stimuli eliciting myeloid differentiation strongly suggest that they play a role in hemopoietic maturation.

The IFN-inducible nucleoprotein IFI 16 is expressed in cells of the monocyte lineage, but is rapidly and markedly down-regulated in other myeloid precursor populations.

IFI 16 is an interferon-inducible nucleoprotein expressed by human monocytes. IFI 16 and a related mouse protein, p202, control cellular proliferation by binding and modulating the functions of cell cycle regulatory factors including p53 and the retinoblastoma gene product, pRb. In this study, we examined IFI 16 expression in myeloid precursor cells cultured in vitro in colony-forming assays using granulocyte (G-) and granulocyte-macrophage (GM-) colony-stimulating factor (CSF). IFI 16 was expressed in 100% of CD34+ cells isolated from human bone marrow. When the CD34+ cells were induced to differentiate, two sub-populations of cells were identified by two-color cytofluorography: the CD14+ (monocytoid) cells all expressed IFI 16, whereas the CD14- (polymorphonuclear precursor) cells did not. The strongest expression of IFI 16 was in the cells staining brightest for CD14, whereas depletion of CD14+ monocytoid cells from mixed monocytic/granulocytic cultures largely abolished IFI 16-stained cells. Furthermore, in eight independent colony-forming assays, the number of IFI 16+ cells correlated closely with the numbers of monocyte precursors identified morphologically (R2 = 0.99), but was unrelated to the numbers of myelocytes, promyelocytes, and metamyelocytes; nor was IFI 16 expressed by erythroid or eosinophil precursors. We conclude that IFI 16 is expressed in CD34+ and monocytoid daughter cells, but is rapidly and markedly down-regulated at the corresponding stages of polymorphonuclear and erythroid development. This differential expression of IFI 16 in myeloid precursor subpopulations and its perceived molecular properties are consistent with a possible role in regulating myelopoiesis.

Induction of phenazine biosynthesis in cultures of Pseudomonas aeruginosa by L-N-(3-oxohexanoyl)homoserine lactone.

A range of Pseudomonas spp. and other Gram-negative bacteria were screened for induction of antimicrobial activity in response to the autoregulatory factor L-N-(3-oxohexanoyl)homoserine lactone. In one of these, P. aeruginosa ATCC 10145, the production of phenazine metabolites was shown to be inducible in a dose-dependent manner. The production of phenazine-1-carboxamide increased over 50-fold compared to control cultures when supplemented with 200 micrograms/ml of the autoregulator. In addition, the production of an unidentified polar antibacterial substance by this strain increased with autoregulator concentration.

Restoration of potassium-stimulated respiration of glycerol-treated muscle.

Agent that produced contracture in skeletal muscle, such as caffeine or K-depolarization, also caused an increased rate of oxygen consumption. Both of these functions are calcium dependent. In this study the respiratory response to K-depolarization and to caffeine was monitored in glycerol-treated and normal frog sartorius muscles. Although glycerol-treated muscle does not contract in response to K-depolarization, it does develop normal caffeine contractures. The respiratory response to both potassium and caffeine is greatly inhibited in glycerol-treated muscles. Pretreatment with dibutyryl cyclic AMP restored the respiratory response to normal levels in glycerol-treated muscle. Pretreatment with low levels of caffeine that had no effect on oxygen uptake markedly enhanced oxygen uptake with higher concentrations of caffeine and resulted in a normal respiratory response to K-depolarization even though there was no tension development. Caffeine had no effect on adenyl cyclase activity even at concentrations that markedly stimulated oxygen uptake. The data suggest that potassium stimulation of oxygen uptake in glycerol treated muscle is uncoupled by a defect in the formation of a cyclic nucleotide cofactor, rather than a defect in calcium influx.

Diffusion measurement in phantoms and tissues using SLIM localization.

A new approach to efficient localized diffusion measurements has been developed and evaluated on phantoms and isolated tissues. The combination of a diffusion-sensitive pulse sequence with SLIM (spectral localization by imaging) makes efficient and accurate localized water and metabolite diffusion measurements possible with a substantial improvement in spatial or time resolution compared to standard methods. Phantom experiments showed that diffusion of substances present in relatively low concentration within small compartments can be measured accurately by this method, suggesting potential applications for diffusion measurements of metabolites in vivo. Experiments on excised rat uterine horns demonstrated the ability of this method to measure localized diffusion of water within irregularly shaped regions of biological samples. Accurate diffusion measurements were achieved in the localized regions with acquisition times less than would have been required by standard diffusion imaging methods.

Quantitative analysis of metabolite levels in normal human subjects by 31P topical magnetic resonance.

We have shown that 31P topical magnetic resonance can be calibrated to obtain quantitative measurements of metabolite levels and intracellular pH in limb muscles of normal human subjects. In some critical situations, TMR yields more accurate results than those obtained by chemical analysis of tissue biopsies.

Novel squalestatins produced by biotransformation.

Microorganisms were screened for the ability to modify the squalene synthase inhibitor squalestatin 1. Biotransformation of 1 by two actinomycetes, S15106 and S15138, yielded three products hydroxylated on the 4,6-dimethyl-oct-2-enoyl side chain either at the 6 position (5) or 7 position (4 two diastereoisomers), and lacking the acetyl ester from the C-1 side chain. Many strains were found to hydrolyse the 4,6-dimethyl-oct-2-enoyl or acetyl esters to yield squalestatins 2 or 3. The 3-methyl ester (6) of 1 was obtained using Fusarium sp. F13945. This fungus also produced a farnesoic acid derivative, possibly in response to inhibition of its squalene synthase by 1. The biotransformation products of 1 all retained potent squalene synthase inhibitory activity.

The squalestatins, novel inhibitors of squalene synthase produced by a species of Phoma. IV. Preparation of fluorinated squalestatins by directed biosynthesis.

Feeding of fluorinated benzoic acids to fermentations of a Phoma sp. resulted in the biosynthesis of a series of novel fluorinated squalestatins. The feeding studies, isolation, structural elucidation and biological activities of these compounds are reported.

The production of novel sordarin analogues by biotransformation.

The biotransformation of the fungal protein synthesis inhibitor sordarin is reported. Nine taxonomically diverse organisms supported the isolation and identification of twelve modified products. The structural diversity of the biotransformation products observed and their value in supporting further chemistry is discussed.

The squalestatins, novel inhibitors of squalene synthase produced by a species of Phoma. I. Taxonomy, fermentation, isolation, physico-chemical properties and biological activity.

During the screening of fungi for inhibitors of squalene synthase, Phoma sp. C2932 was found to produce three structurally related novel inhibitors. These compounds, designated the squalestatins, exhibited potent activity against both mammalian (rat liver) and fungal (Candida albicans) squalene synthase. Furthermore, they also had broad spectrum in vitro antifungal activity.

Novel inhibitors of fungal protein synthesis produced by a strain of Graphium putredinis. Isolation, characterisation and biological properties.

The isolation and structure determination of 6 analogues of the fungal protein synthesis inhibitor GR135402, from Graphium putredinis, is described. The relative potencies of the compounds as protein synthesis inhibitors and as in vitro antifungal agents provide interesting insights into the structure-activity relationships in this series.

Isolation and characterisation of an antifungal antibiotic (GR135402) with protein synthesis inhibition.

A novel antifungal antibiotic GR135402 has been isolated from a fermentation broth of Graphium putredinis which inhibited protein synthesis in Candida albicans but not rabbit reticulocytes. The spectrum of activity included C. albicans and Cryptococcus neoformans but not some other Candida species or Aspergillus species. Therapeutic efficacy in a mouse model of systemic candidosis was attained following parenteral dosing.

The suitability of carboplatin solutions for 14-day continuous infusion by ambulatory pump: an HPLC-dynamic FAB study.

The stability of aqueous carboplatin solutions over 14 days has been studied at 37 and 60 degrees C. High-performance liquid chromatography and dynamic FAB mass spectrometry studies have shown that carboplatin solutions were stable at 37 degrees C but degraded at 60 degrees C. Fluid loss through evaporation was significant at the higher temperature.

Resolution of 4-amino-cyclopentanecarboxylic acid methyl esters using hydrolytic enzymes.

A number of esterases (EC 3.1.1.1) and lipases (EC 3.1.1.3) of microbial and mammalian origin were screened for the ability to resolve racemic 4-amino-cyclopentanecarboxylic acid methyl ester derivatives as potential intermediates in the production of carbocyclic nucleosides. Surprisingly, functionalization of the remote amino group had a profound effect on both the rate and enantioselectivity of hydrolysis of the methyl ester. 4-(Benzoylamino)-2-cyclopentenecarboxylic acid, methyl ester (V) with pig liver esterase gave the highest enantioselectivity. The residual ester, which was of the correct absolute stereochemistry [(+) 1S, 4R] for carbocyclic nucleoside synthesis, could be obtained in high optical purity. Optimization of pH, solvent type, and concentration improved the enantioselectivity of the process by a further twofold.

An efficient process for production of N-acetylneuraminic acid using N-acetylneuraminic acid aldolase.

N-acetyl-D-neuraminic acid (Neu5Ac) aldolase (EC 4.1.3.3) has bee reported for synthesis of Neu5Ac,1-5 but there are no reports of processes which do not have significant drawbacks for large-scale operation. Here, Neu5Ac aldolase from an overexpressing recombinant strain of Escherichia coli has been used to develop an immobilized enzyme process for production of Neu5Ac. The enzyme was immobilized onto Eupergit-C and could be reused many times in the reaction. Base-catalyzed epimerization of N-acetyl-D-glucosamine (GlcNAc) yielded GlcNAc/N-acetyl-D-mannosamine (ManNAc) mixtures (c 4:1) which could be used directly in the aldolase reaction; however, inhibition of the enzyme by GlcNAc limited the concentration of ManNAc which could be used in the reaction by this approach. This necessitated the addition of a large molar excess of pyruvate (five- to seven-fold) to drive the equilibrium over to Neu5Ac; nevertheless, a method has been developed to remove the excess pyruvate effectively by complexation with bisulfite, thus allowing Neu5Ac to be recovered by absorption onto an anion-exchange resin. In a second approach, a method has been developed to enrich GlcNAc/ManNAc mixtures for ManNAc. ManNAc can be used at high concentrations in the reaction, thus obviating the need to use a large molar excess of pyruvate. Neu5Ac can be isolated from such reaction mixtures by a simple crystallization. This work shows the importance of integrated process solutions for the effective scale-up of biotransformation reactions.

Enzymatic production of optically pure (2'R-cis)-2'-deoxy-3'-thiacytidine (3TC, lamivudine): a potent anti-HIV agent.

Although equipotent in terms of antiviral activity, the two enantiomers of 2'-deoxy-3'-thiacytidine (BCH 189) differ markedly in their cytotoxicity. (2'R-cis)-2'-deoxy-3'-thiacytidine (3TC) is substantially less toxic than its optical antipode, and is undergoing development for the therapy of HIV infection. Cytidine deaminase from Escherichia coli is shown here to deaminate 2'-deoxy-3'-thiacytidine enantioselectively to leave 3TC essentially optically pure. This reaction has been used to develop a process for production of 3TC in multikilogram amounts. The production of cytidine deaminase was enhanced by strain improvement, fermentation development, and finally by cloning and overexpression of the gene. The enzyme was immobilized on Eupergit-C, which allowed it to be reused many times. The biotransformation conditions were optimized so that the best use could be made of the catalyst. A robust scaleable product isolation process was developed to yield the crystalline product. Overall, yields through the resolution process of 76% were obtained. All aspects of this process are capable of substantial further scaleup with only minor modifications.