A 7-day intravenous toxicity study and neurotoxicity assessment of pyridorin in Sprague-Dawley rats.

Pyridorin®, a naturally occurring metabolite of vitamin B6 that inhibits and scavenges reactive oxygen species, is being developed as a potential therapeutic for acute kidney injury. An investigational new drug application (IND) was opened for Pyridorin in support of its ongoing oral drug clinical development program. Currently, a Pyridorin intravenous (IV) formulation is being developed for use in surgical patients. To support the IND for Pyridorin, a full battery of nonclinical Good Laboratory Practice compliant studies was performed with no neurological or behavioral signs of toxicity seen following oral or IV administration of pyridoxine dihydrochloride (the active ingredient in Pyridorin). However, excessive ingestion of vitamin B6 has been reported to cause neurotoxic syndrome in humans. Therefore, under Food and Drug Administration recommendation, a 7-day IV study in rats was conducted to further evaluate the drug's potential to cause neurotoxicity. Blood plasma samples indicated that exposure to pyridoxamine dihydrochloride and its metabolites, pyridoxal, pyridoxine, and 4-pyridoxic acid was linearly dose proportional and independent of gender. At doses of up to 200 mg/kg/day pyridoxine dihydrochloride, no treatment-related effects were seen in rats, providing further evidence for the absence of pyridoxine dihydrochloride-related changes in the nervous system. A no observed adverse effect level of 200 mg/kg/day was identified for this study.

Technical issues in using robots to reproduce joint specific gait.

Reproduction of the in vivo motions of joints has become possible with improvements in robot technology and in vivo measuring techniques. A motion analysis system has been used to measure the motions of the tibia and femur of the ovine stifle joint during normal gait. These in vivo motions are then reproduced with a parallel robot. To ensure that the motion of the joint is accurately reproduced and that the resulting data are reliable, the testing frame, the data acquisition system, and the effects of limitations of the testing platform need to be considered. Of the latter, the stiffness of the robot and the ability of the control system to process sequential points on the path of motion in a timely fashion for repeatable path accuracy are of particular importance. Use of the system developed will lead to a better understanding of the mechanical environment of joints and ligaments in vivo.

The effect of digital image resolution and compression on anterior eye imaging.

AIM: To determine the theoretical and clinical minimum image pixel resolution and maximum compression appropriate for anterior eye image storage. METHODS: Clinical images of the bulbar conjunctiva, palpebral conjunctiva, and corneal staining were taken at the maximum resolution of Nikon:CoolPix990 (2048x1360 pixels), DVC:1312C (1280x811), and JAI:CV-S3200 (767x569) single chip cameras and the JVC:KYF58 (767x569) three chip camera. The images were stored in TIFF format and further copies created with reduced resolution or compressed. The images were then ranked for clarity on a 15 inch monitor (resolution 1280 x 1024) by 20 optometrists and analysed by objective image analysis grading. Theoretical calculation of the resolution necessary to detect the smallest objects of clinical interest was also conducted. RESULTS: Theoretical calculation suggested that the minimum resolution should be > or = 579 horizontal pixels at 25x magnification. Image quality was perceived subjectively as being reduced when the pixel resolution was lower than 767 x 569 (p<0.005) or the image was compressed as a BMP or <50% quality JPEG (p<0.005). Objective image analysis techniques were less susceptible to changes in image quality, particularly when using colour extraction techniques. CONCLUSION: It is appropriate to store anterior eye images at between 1280 x 811 and 767 x 569 pixel resolution and at up to 1:70 JPEG compression.

Continuous-fiber preform reinforcement of dental resin composite restorations.

OBJECTIVES: Direct-filling resin composites are used in relatively small restorations and are not recommended for large restorations with severe occlusal-stresses. The aim of this study was to reinforce composites with fiber preforms, and to investigate the effects of layer thickness and configurations on composite properties. It was hypothesized that fiber preforms would significantly increase the composite's flexural strength, work-of-fracture (toughness) and elastic modulus. METHODS: Glass fibers were silanized, impregnated with a resin, cured, and cut to form inserts for tooth cavity restorations. Also fabricated were three groups of specimens of 2mm x 2mm x 25 mm: a fiber preform rod in the center of a hybrid composite; a thin fiber layer on the tensile side of the specimens; and a thin fiber layer sandwiched in between layers of a hybrid composite. These specimens were tested in three-point flexure to measure strength, work-of-fracture and modulus. Optical and scanning electron microscopy were used to examine the restorations and the fiber distributions. RESULTS: Microscopic examinations of insert-filled tooth cavities showed that the fibers were relatively uniform in distribution within the preform, and the inserts were well bonded with the surrounding hybrid composite. Specimens consisting of a fiber preform rod in the center of a hybrid composite had a flexural strength (mean (SD); n=6) of 313 (19)MPa, significantly higher than 120 (16)MPa of the hybrid composite without fibers (Tukey's at family confidence of 0.95). The work-of-fracture was increased by nearly seven times, and the modulus was doubled, due to fiber preform reinforcement. Similar improvements were obtained for the other two groups of specimens. SIGNIFICANCE: Substantial improvements in flexural strength, toughness and stiffness were achieved for dental resin composites reinforced with fiber preforms. The method of embedding a fiber preform insert imparts superior reinforcement to restorations and should improve the performance of direct-filling resin composites in large restorations with high occlusal-loads.

Hydrolytic degradation of tyrosine-derived polycarbonates, a class of new biomaterials. Part II: 3-yr study of polymeric devices.

The kinetics and mechanisms of in vitro degradation of tyrosine-derived polycarbonates, a new class of polymeric biomaterials, were studied extensively at 37 degrees C. These polymers carry an alkyl ester pendent chain that allows the fine-tuning of the polymer's material properties, its biological interactions with cells and tissue, and its degradation behavior. The polymer carrying an ethyl ester pendent chain, poly(DTE carbonate), has been established as a promising orthopedic implant material, exhibiting bone apposition when in contact with hard tissue. Tyrosine-derived polycarbonates are relatively stable and degrade only very slowly in vitro. Therefore, accelerated studies were conducted at 50 and 65 degrees C to observe the behavior of polymers during the later stages of degradation. Varying the pendent chain length affected the rate of water uptake, initial degradation rate, and physical stability of the polymeric devices. During the 3-yr study, the polymer degraded by random chain cleavage of the carbonate bonds, accompanied by a relatively small amount of pendent chain de-esterification. No mass loss was observed during this period at 37 degrees C, but mass loss was readily evident during the accelerated studies at 50 and 65 degrees C. Thus, it is reasonable to assume that mass loss will occur also at 37 degrees C, albeit only after extensive backbone carbonate cleavage and pendent chain ester hydrolysis. The dimension and surface area of the devices influenced the initial degradation rate, but did not significantly affect the overall rate of degradation. No evidence of "acid dumping" or the release of acidic residues found during the degradation of poly(D,L-lactic acid) were observed for this family of tyrosine-derived polycarbonates.

Beating heart surgery: why expect less central nervous system morbidity?

BACKGROUND: The incidence and etiology of brain dysfunction after conventional coronary artery bypass surgery using cardiopulmonary bypass (CPB) are reviewed. METHODS: Stroke rates and incidences of cognitive dysfunction from various studies are considered. Mechanisms of injury including cerebral embolization as detected by transcranial Doppler and retinal angiography, and imaging-based evidence for postoperative cerebral edema, are discussed. Preliminary results from a prospective clinical trial assessing cognitive dysfunction after beating heart versus conventional coronary artery bypass with CPB are discussed. RESULTS: Initial evidence for lower overall postoperative morbidity, and for a lower incidence of cognitive dysfunction specifically, after nonpump coronary revascularization is presented. CONCLUSIONS: Beating heart surgery results in less potential for generation of cerebral emboli and appears to produce a lower incidence of cognitive dysfunction in both short- and intermediate-term postoperative follow-up periods as compared with conventional coronary artery bypass surgery using CPB.

Guamanian neurodegenerative disease: electrophysiologic findings.

Amyotrophic lateral sclerosis (ALS), parkinsonism and/or dementia are highly prevalent among the Chamorro population of Guam. The incidence of Guamanian ALS has markedly declined in recent years, but these incidence figures may reflect underascertainment of subclinical disease. Guamanian Chamorro patients have not been systematically studied using modern clinical neurophysiological techniques. Electromyography (EMG: needle exam and nerve conduction studies) was used to study 29 patients with the major subtypes of Guamanian neurodegenerative disease, as well as 11 neurologically normal Guamanian Chamorro subjects. Central conduction was assessed by somatosensory evoked potentials (SEP's) in 16 patients. EMG evidence of peripheral neuropathy, (often subclinical) was found in 45% of Guamanian patients but no Chamorro control subjects. Diabetes mellitus, which is highly prevalent in this population, was present in some, but not all of these cases. Clinically unsuspected motor neuron disease was identified by EMG in only one of the 23 Guamanian patients with parkinsonism and/or dementia and in none of the 11 Chamorro control subjects. Two of seven patients with the clinical phenotype of Guamanian ALS had a more benign EMG pattern on the needle electrode exam with absence of fibrillation and fasciculation potentials. Three of 16 patients (all with parkinsonism and dementia) had mildly abnormal tibial SEP's. No patient had EMG evidence of myopathy or a defect of neuromuscular transmission. We conclude: (1) peripheral neuropathy may be a manifestation of Guamanian neurodegenerative disease; (2) the declining prevalence of ALS on Guam is not associated with the development of a subclinical form of motor neuron disease; (3) the substantial overlap of Guamanian ALS with parkinsonism-dementia reported in prior decades is no longer apparent; (4) abnormal central conduction, as assessed by tibial SEP's, is present in some patients with Guamanian parkinsonism-dementia.

Gaining a competitive edge through HIS technology.

Streamlined procedures, fewer errors, improved consultation on patient care, and lowered costs are among the advantages cited by healthcare facilities that are using information technology in efforts to outpace their competitors. Technology ranging from executive information systems to medical record imaging can provide improved access to data and aid strategic decision making. Even so, the perceived benefits of a particular form of technology should not outweigh sound business planning.

Continuing education needs of board members, administrators, and health care personnel.

Our society is becoming more aware of the basic fact that education is a lifelong process. The dynamic world in which we live requires that we continually update our knowledge and skills. Educational institutions that have traditionally viewed their mission as one of providing a sequence of courses leading to a specific degree are now expanding their role to include postgraduate nondegree offerings. The MIS area certainly ranks high among those fields that have a dynamic characteristic. Institutions with particular academic strength in this area can provide a needed service--and at the same time create a market niche for themselves--by developing and presenting continuing education programs in the area of MIS.

Performance of a longitudinally compliant PTFE vascular prosthesis in an ovine A-V fistula model.

Nine chronic implants of 6 mm diameter vascular grafts were performed in an ovine model to compare the performance of the longitudinally compliant Ultraflex PTFE-Plus vascular prosthesis with that of the Gore-Tex reinforced expanded polytetra fluoroethylene (PTFE) graft. Implants were performed in the sheep neck as right carotid to left jugular arterio-venous (A-V) grafts. Grafts were explanted after 6 months unless they occluded earlier. The Ultraflex graft demonstrated superior handling characteristics during implant. Patency rate at 6 months was 60% (three of five) for the Ultraflex graft (experimental graft by Baxter Edwards, CVS Division, Irvine, CA), and 25% (one of four) for the Gore-Tex graft (WL Gore & Associates, Inc., Flagstaff, AZ). The Gore-Tex prosthesis had more calcification of the graft wall.

Dynamic internal compliance measurements of fresh and fixed artery.

A new device is used to compare dynamic internal compliance of biologic vascular prostheses derived from bovine artery. These grafts were prepared using dialdehyde starch (DS), and a polyepoxy polyether compound (PC), as crosslinking agents. Internal compliance was measured using the Dynatek DCT1 Dynamic Compliance Tester, developed by Dynatek Laboratories. This device utilizes a variable speed DC motor coupled to miniature bellows, which displace a stroke volume controllable to within 0.5 microliter. The bellows displace fluid into and out of the graft, resulting in a sinusoidal change in internal pressure. Volumetric displacement is monitored by a linear voltage displacement transducer (LVDT) mounted on the motor coupling, whereas internal graft pressure is monitored by a miniature pressure transducer mounted on the closed end of the graft. For this series of experiments, a pressure range of 80-120 mmHg was used, with a cycle speed of 80 cycles/min. Preliminary results indicate that PC fixation of bovine artery results in a vascular graft with internal compliance somewhere between fresh tissue and DS fixation. This data confirms qualitative assessment. The DCT1 provides valuable comparative information on the dynamic internal compliance of vascular prostheses. These measurements reflect what occurs in the graft lumen, since they take into account wall compression and axial deformation. Further development is required to separate radial and longitudinal compliance components.

Expression and processing of recombinant human terminal transferase in the baculovirus system.

Overproduction of human terminal transferase protein has now been accomplished by cloning the coding sequence of human terminal transferase into a baculovirus, where the expression of terminal transferase is under the control of the polyhedrin protein promoter. Two constructs were made, one producing a protein containing the entire terminal transferase fused to 12 amino acids from the NH2 terminus of the polyhedrin protein, and the other producing 58-kDa human terminal transferase. The terminal transferase levels expressed in cells infected with either recombinant baculovirus are around 10,000 units/10(7) cells at 48 h postinfection, about 200-fold greater than levels expressed in thymus and cultured lymphoblastoid cells. The chimeric polyhedrin/human terminal transferase protein produced in the infected insect cells has a molecular weight of about 60,000 while the nonfused recombinant human terminal transferase is identical in molecular weight to that present in human lymphoblastoid cells. Both forms of recombinant terminal transferase show immunological and enzymatic activity. When infected cells are pulse-labeled with [35S] methionine at 42-45 h postinfection, about 10% of newly synthesized protein is terminal transferase. Both forms of terminal transferase are phosphorylated in recombinant virus-infected cells as demonstrated by pulse-labeling infected cells with 32P-inorganic phosphate and isolation of labeled terminal transferase peptides by immunoprecipitation.

Evaluation of compliant and noncompliant PTFE vascular prostheses.

Four chronic implants of 6 mm diameter grafts compare a new longitudinally compliant PTFE-composite vascular prosthesis (ULTRAFLEX PTFE-PLUS) with clinical grade microporous expanded polytetrafluoroethylene (PTFE). Implants were carried out in the ovine carotid-jugular position as A-V fistulae. All four grafts were subjected to sham dialysis punctures in identical fashion with 14-gauge needles. After 60 days the grafts were explanted and submitted for histologic evaluation. All four grafts were patent at explant. The compliant PTFE (ULTRAFLX PTFE-PLUS) lumen surface was thrombus free. Neointima lining at the proximal and distal anastomoses was similar in both test and controls. Tissue ingrowth and outer capsule formation were virtually identical. Foreign body reaction was minimal. Superior handling and self-sealing properties distinguished test from controls. Long term animal studies are in progress for vascular access and peripheral applications.

Nitrous oxide and human state-dependent memory.

State-dependent effects of nitrous oxide on human memory were examined by administering serial and paired-associate learning tasks to subjects receiving 20 and 30% nitrous oxide or placebo. Nitrous oxide in 30% concentration impaired learning of both tasks. In addition, it produced an atypical form of asymmetric state-dependent memory; subjects who learned while receiving placebo and recalled while receiving nitrous oxide displayed the worst recall.

Prediction of the frequencies of restriction endonuclease recognition sequences using di- and mononucleotide frequencies.

The calculation of probabilities of nucleotide sequences from the frequencies of dinucleotides is described. The dinucleotide and mononucleotide frequencies used can be obtained from nearest neighbor analysis or from databank sequences. If dinucleotide and mononucleotide frequencies from nearest neighbor analysis are used, probabilities for oligonucleotides can be calculated for genomes in which there is little or no sequence data. Within a given genome, a broad range of probabilities for hexanucleotide palindromes with the same base composition is predicted and shown (14).

Sequence and transcription of tRNAVal gene from Xenopus laevis.

A DNA fraction enriched in tRNA genes has been prepared by CsCl density gradient centrifugation of Xenopus laevis DNA in the presence of actinomycin D. This DNA fraction was cut with the restriction endonuclease EcoRI and the fragments 800-900 base pairs in size were cloned into the plasmid pBR325. Recombinant DNAs were screened by hybridization to labeled tRNA and for the ability to support transcription in vitro. The entire sequence of one fragment was determined by sequencing the ends of an overlapping set of deletion fragments. A sequence homologous to tRNAVal from mammalian sources was found in this fragment and it was shown that this sequence corresponds to the region of the fragment that is transcribed. The cloned fragment was also transcribed in vivo after injection into X. laevis oocytes. The RNA that was synthesized in the oocytes was digested with ribonuclease T1 and the oligonucleotides were separated to produce a two-dimensional fingerprint. The results of the analysis of the oligonucleotides are consistent with the sequence determined for the tRNAVal gene. The X. laevis genome has 200-250 copies of the 892 base pair EcoRI fragment and additional copies of a 4100 base pair EcoRI fragment that each contain a tRNAVal gene. Digestion of X. laevis DNA with several other restriction endonucleases reveals that the cloned fragment that contains the tRNAVal gene is part of a longer sequence element that is tandemly repeated in the genome.

Expression of human terminal deoxynucleotidyl transferase in Escherichia coli.

A cloned DNA fragment related to pT17 containing a partial cDNA sequence of human terminal deoxynucleotidyl transferase was used as a probe to screen for the full length cDNA sequence of the enzyme in a lambda gt11 library constructed from human lymphoblastoid KM-3 cDNA. A recombinant containing a 2068-base pair insert was isolated and recloned into the EcoRI site of the sequencing plasmic pUC-8 as two subclones, pT711 and pT106. DNA sequencing and hybridization studies showed that pT711 contains the pT17 sequence and an additional 172 upstream nucleotides. pT711 represents the coding sequence for the carboxyl half of the terminal transferase protein. pT106, containing a 965-base pair insert, hybridizes to the same mRNA as pT711 on Northern blots and contains an open reading frame that is in phase with the reading frame of the insert in pT711. Amino acid sequencing of the 58-kDa peptide of the calf thymus terminal transferase failed, indicating that the N terminus is blocked. N-Terminal sequencing of a 56-kDa form of the protein produced 24 amino acids corresponding to the translated human cDNA coding sequence starting at residue 398 of the insert in pT106 with 83% homology between bovine and human sequence. The initiation codon is assigned to an ATG sequence at nucleotide 329 of the insert in pT106. Comparison of the translated human terminal transferase sequence with peptides from the calf thymus enzyme showed that the homology between the human and bovine enzyme is better than 90% among 263 amino acids determined. The coding sequences in pT106 and pT711 were recloned into an expression plasmid pUC-19 downstream from the lac promoter and in phase with the coding sequence of the lac Z gene. Lysates of bacteria carrying the reconstructed coding sequence of human terminal transferase contain a fused protein of 60 kDa that reacts with rabbit antibody to terminal transferase on immunoblots and exhibits enzyme activity. Isolation of this fused protein from bacterial lysates with mouse monoclonal antibody to human terminal transferase produces the expected protein of 60 kDa.

Chromosome localization of the gene for human terminal deoxynucleotidyltransferase to region 10q23-q25.

Complementary DNA clones representing the 3' half, the 5' half, and the entire coding region of the human terminal deoxynucleotidyltransferase gene (TdT; DNA nucleotidylexotransferase, nucleosidetriphosphate: DNA deoxynucleotidylexotransferase, EC 2.7.7.31) were used to screen a panel of mouse X human somatic cell hybrid DNAs to determine the chromosomal location of the human TdT gene. The results of the Southern transfer analysis of hybrid DNAs indicate that the gene for TdT is located on human chromosome 10. The in situ hybridization technique was then used to further localize the gene for TdT to region q23-q25 of human chromosome 10.