Mechanism-Based Inhibitors: Development of a High Throughput Coupled Enzyme Assay to Screen for Novel Antimalarials.

Identifying potent enzyme inhibitors through a robust HTS assay is currently thought to be the most efficient way of searching for lead molecules. We have developed a HTS assay that mimics a crucial step in an essential metabolic pathway, the purine salvage pathway of the malarial parasite Plasmodium falciparum. In this assay we have used purified recombinant enzymes: hypoxanthine guanine phosphoribosyl transferase (HGPRT) and inosine monophosphate dehydrogenase (IMPDH) from the malarial parasite and the human host, respectively. These two enzymes, which work in tandem, are used to set up a coupled assay that is robust enough to meet the stringent criteria of an HTS assay. In the first phase of our screen we seem to have identified novel inhibitors that kill the parasite by inhibiting the salvage pathway of the parasite.

Analytic model to predict the strength of tendon repairs.

We developed an analytic model to predict suture load-sharing immediately after flexor tendon repair in the hand. Tendon repair was mathematically modeled as two nonlinear springs in parallel, representing separate core and peripheral sutures that were in series with a third nonlinear spring representing the tendon. To serve as a basis for, and validation of, our analytic model, fresh human flexor digitorum profundus tendons were harvested and mechanically tested either intact or after surgical repair in a variety of ways: core suture alone, superficial peripheral suture alone, deep peripheral suture alone, core suture plus superficial peripheral suture, and core suture plus deep peripheral suture. The stiffness and strength of the composite repairs predicted with use of the analytic model were comparable with those determined experimentally. Furthermore, the model predicted inequities in suture load-sharing, with 64% of the applied load carried by the peripheral suture when it was placed superficially, as compared with 77% when the peripheral suture was placed deep. Our results demonstrate a disparity in load-sharing within composite suture systems, the rectification of which may lead to significant improvement in the repair strength. To this end, we expect that our analytic model will serve as a basis for the design of more efficient, and consequently stronger, suture techniques.

Boron-containing polyamines as DNA targeting agents for neutron capture therapy of brain tumors: synthesis and biological evaluation.

Three series of new boron-containing spermidine/spermine (SPD/SPM) analogues have been synthesized: N1- and N5-(4-carboranylbutyl) SPD/SPM derivatives (SPD-1, SPD-5, SPM-1, SPM-5); N1,N10-diethyl-N5-(4-carboranylbutyl)spermidine (DESPD-5), N1,N14-diethyl-N5-(4-carboranylbutyl)spermine (DESPM-5); and N5,N10-bis(4-carboranylbutyl)spermine (SPM-5,10). In vitro studies using rat F98 glioma cells have shown that these polyamines retain the ability to displace ethidium bromide from calf thymus DNA and are rapidly taken up by F98 glioma cells. However, their cytotoxicities, especially those with terminal N-substituted (SPD-1, SPM-1) boron compounds, are greater than those of SPD/SPM. Nevertheless, the groundwork has been created for a new class of boron-containing compounds that maybe useful for boron neutron capture therapy of tumors.

Partial lacerations of human digital flexor tendons: a biomechanical analysis.

The biomechanical properties of human flexor tendons with partial lacerations have not been previously studied. To determine the loss of tensile strength with varying degrees of partial laceration, tensile tests were performed on 2 matched groups of human cadaver flexor tendons: One group had 50% while the other had 75% transverse volar lacerations of the anteroposterior diameter. The mean failure load of the 50%-lacerated tendons was 93% higher than that of the 75%-lacerated tendons. The forces tolerated by the lacerated tendons before failure were also compared to those measured in vivo during physiologic loading. The breaking loads of both 50%- and 75%-lacerated tendons far exceeded the in vivo forces measured in human flexor tendons during unresisted active finger movement (up to 34 N). Further, the breaking loads of 50% lacerations was higher than the in vivo forces during resisted active finger movement (up to 117 N). This study demonstrates that the threshold load levels to rupture of 50% and 75% lacerations are higher than physiologic load levels measured during active motion, suggesting that partial flexor tendon lacerations of up to 75% can withstand in vivo forces associated with active unresisted mobilization of the digital flexor tendon.

Frozen storage affects the compressive creep behavior of the porcine intervertebral disc.

STUDY DESIGN: A biomechanical study of the compressive creep behavior of the porcine intervertebral disc before and after frozen storage. OBJECTIVE: To determine whether frozen storage alters the creep response, hydration, and nuclear swelling pressure of the intact intervertebral disc. SUMMARY OF BACKGROUND DATA: The mechanical response of the disc is dominated by swelling and fluid flow, whose effects are time-dependent. Because fluid content, which may change during storage, plays a significant role in the disc's time-dependent behavior, changes in mechanical response due to freezing may have been missed in previous studies that focused on time-independent behavior only. METHODS: Porcine intervertebral discs were tested in repeated cycles of compressive creep either immediately postmortem or after 3 weeks of frozen storage. Swelling pressure and nuclear hydration were also measured in fresh and frozen discs. A fluid transport model was used to analyze the creep data. RESULTS: The creep behavior of the intact porcine intervertebral disc is dramatically affected by frozen storage. The apparent permeability of the frozen discs was 82% higher than that of the fresh discs, and the swelling pressure of frozen discs was 25% lower in frozen discs (P < 0.01). The behavior of fresh and frozen discs became more dissimilar with repeated cycles of creep. CONCLUSIONS: In vitro tests of frozen porcine intervertebral discs do not represent fresh behavior. Frozen storage appears to permanently alter disc behavior. The precise nature of any freezing-induced damage, and whether frozen storage similarly affects human discs, remains to be seen.

Alternative forms of the human thioredoxin mRNA: identification and characterization.

Thioredoxin (TRX) is an ubiquitous and relatively conserved oxidoreductant enzyme which is involved in a multitude of redox reactions through the formation of reversible disulfide bonds. A recent report indicates the presence of novel isoforms of TRX proteins isolated from MP6 cell lines [Rosén et al., Int. Immunol. 7 (1995) 625-633]. In these isoforms, as evidenced from amino acid sequencing, several Lys residues of the wild-type sequence were replaced by Arg. Although the human genome contains several (isoformic) copies of the TRX gene, only one appears to be transcriptionally active [Kaghad et al., Gene, 140 (1994) 273-278]. As we characterized the isoforms of TRX mRNAs, we found that several MP6 TRX cDNA clones were devoid of the characteristic poly(A) tail. In order to increase the efficiency of isolating mRNAs without the poly(A) tail, we developed a novel procedure for exclusive capturing of a specific mRNA by magnetic beads coated with biotinylated antisense oligodeoxyribonucleotide. Using this method on MP6 cell total RNA, we isolated an additional truncated version of the TRX mRNA. This latter form does not produce any variant TRX enzyme, as an inframe stop codon truncates the product. This isoform was also present in mRNAs isolated from human placenta, leucocyte cells and Molt4 cells, the latter two being the progenitors of MP6 cells. From a thorough analysis of the sequence of the truncated TRX mRNA, we conclude that this variant originated from an event of altered splicing, as consensus splice sites were present in the normal TRX cDNA at precise positions.

Effect of UVB radiation on corneal aldehyde dehydrogenase.

PURPOSE: A Class 3 aldehyde dehydrogenase happens to be a major soluble protein constituent of the cornea. Its role is conjectured to be manifold: to protect the tissue from oxidative damage by eliminating the toxic aldehydes produced upon lipid peroxidation under oxidative stress, to act as an UV-absorber, and to maintain the level of the coenzyme NADH in the cornea. We have studied the effect of UVB on the structure and enzyme activity of corneal aldehyde dehydrogenase. METHODS: Aldehyde dehydrogenase was irradiated at 295 nm for varying periods of time and change in its enzyme activity assayed. The structural changes in the molecule accompanying irradiation were monitored using fluorescence and circular dichroism spectroscopy, and its hydrodynamic behavior and surface hydrophobicity studied using gel filtration chromatography and binding of the hydrophobic fluorophore ANS. The protective ability of aldehyde dehydrogenase in preventing aggregation of photolabile proteins, such as Gamma-crystallin of the eye lens, was studied by monitoring the scattering value of the test protein with irradiation by UVB. RESULTS: Aldehyde dehydrogenase is seen to undergo photodamage with alterations in its quaternary structure, though no significant change is noticed in the peptide chain conformation. Under such conditions the molecule continues to act as a protectant by preventing aggregation of photolabile proteins such as the eye lens Gamma-crystallin. CONCLUSIONS: Our earlier studies have shown that the free sulfhydryl groups are important for the antioxidant abilities of aldehyde dehydrogenase. Its protective ability towards photoaggregation of Gamma-crystallin seen here might arise both due to: (i) oxyradical quenching and (ii) the increased surface hydrophobicity of the molecule upon irradiation, which allows it to bind to, and thus inhibit the aggregation of interacting proteins.

Effect of peripheral suture depth on strength of tendon repairs.

The peripheral suture in flexor tendon repairs was originally introduced to "tidy up" the repair site and to improve gliding after core suture placement. More recently, it has been proposed that the peripheral suture can be an important structural component of tendon repair; yet, the effect of peripheral suture technique as an independent factor on the strength of the tendon repair has had little attention. We proceeded to determine whether differing levels of penetration of the tendon by the peripheral suture significantly affect the strength of the repair. We performed tensile tests on two groups of fresh frozen cadaver flexor digitorum superficialis tendons that had lacerations repaired by modified Kessler core sutures: one group had supplemental superficial peripheral sutures while the other group had supplemental deep (half the depth to the center of the tendon) peripheral sutures. The mean failure load of the deep peripheral suture group (38.96 N) was almost 80% greater than that of the superficial suture group (21.68 N). This increased strength may be due to an improved tendon-suture interface, with the deep peripheral suture acting as an auxiliary core suture. Our results with deep peripheral sutures, a simple technique based on a modification of commonly employed suture patterns, demonstrate substantially improved repair strength. This suture has the potential to be used in conjunction with early active finger motion rehabilitation program after flexor tendon repair.

Comparative mechanical analysis of dorsal versus palmar placement of core suture for flexor tendon repairs.

This study was designed to evaluate the effect of core suture placement in the coronal plane on the tensile strength of flexor tendon repairs. We compared the tensile strength of modified Kessler core sutures placed in the palmar versus the dorsal side of matched-lacerated human cadaver flexor digitorum profundus tendons tested in vitro under static loading. We also compared the relative strengths of the dorsal and palmar portions of the flexor digitorum tendons. The mean failure load of a dorsal-side modified Kessler suture was 26.5% greater than that of a palmar-side modified Kessler suture. The mean strength of the dorsal half of the flexor digitorum tendons was 58.3% greater than that of the palmar half of the flexor digitorum tendons. Our data demonstrate that the dorsal tendon can sustain greater loads-to-failure than the palmar tendon. This suggests that there are biomechanical advantages to dorsal as opposed to palmar placement of the core suture in clinical tendon repair.

Hickman catheter implantation in the treatment of acute leukaemia.

Between June 1984 and May 1986, 13 Hickman catheters were introduced in 11 patients for the treatment of acute leukaemia. The catheters remained in situ for a mean period of 77 days (range 1-180). Two of the patients developed haematomas at the entry site and one patient had a blockage of the catheter due to a blood clot which required intervention. Six patients had documented bacteraemia which on blood culture showed enteric organisms and was not catheter related. No catheter had to be removed due to a catheter-related complication. Hickman right atrial catheter provides a safe and reliable venous access with minimum complications and is well tolerated by the patient.