In vitro glycation of human serum albumin by dihydroxyacetone and dihydroxyacetone phosphate.

Amino groups in proteins can non-enzymatically react with reducing sugars to generate a structurally diverse group of compounds referred to as advanced glycation end products (AGEs). The in vivo formation of AGEs contributes to some of the complications of diabetes including atherosclerosis, cataract formation, and renal failure. The formation of AGEs is dependent on both sugar and protein concentrations. Increases in temperature, pH, and exposure time of sugars to the proteins also play a significant role in the rate of AGE formation. This study focuses on the use of a combination of analytical techniques to study the in vitro AGE formation of HSA with dihydroxyacetone phosphate (DHAP), a ketose generated during glycolysis, and its dephosphorylated analog, dihydroxy acetone (DHA), commonly used as a browning reagent in skin tanning preparations. The extent of AGE formation was affected by DHAP and DHA concentrations and by the duration of HSA exposure to these glycating agents. Increases in temperature and pH sped the glycation process and enhanced the formation of the AGEs of HSA. MALDI-TOF mass spectroscopic data provided a reliable result to evaluate the extent of the AGE formation.

The in vitro glycation of human serum albumin in the presence of Zn(II).

Amino groups of human serum albumin (HSA) can react non-enzymatically with carbonyl groups of reducing sugars to form advanced glycation end products (AGEs). These AGEs contribute to many of the chronic complications of diabetes including atherosclerosis, cataract formation and renal failure. The current study focused on in vitro non-enzymatic reactivity of glyceraldehyde (GA) and methylglyoxal (MG) with HSA and evaluated the rate and extent of AGE formation in the presence of varied concentrations of Zn(II). At normal physiological conditions, GA and MG readily react with HSA. The presence of Zn(II) in HSA-GA or HSA-MG incubation mixtures reduced AGE formation. This finding was confirmed by UV and fluorescence spectrometry, HPLC techniques, and matrix assisted laser desorption ionization mass spectrometry (MALDI-TOF). HPLC studies revealed decreased adduct formation of the glycated protein in the presence of Zn(II). The inhibition of AGE formation was intense at elevated Zn(II) concentrations. The results of this study suggest that Zn(II) may prove to be a potent agent in reducing AGE formation.

Neural network analysis of employment history as a risk factor for prostate cancer.

BACKGROUND: Prostate cancer is the most common non-cutaneous malignancy in men. Its etiology likely involves environmental exposures and demographic factors. OBJECTIVE: Investigate the potential relationship between occupation history and prostate cancer risk in a population-based, case-control study (n=1365). METHODS: The variables: race, age group, smoking status, income, marital status, education and the first 15 years of employment history were examined by sequential odds ratio analysis then compared to a neural network consensus model. RESULTS: Both the sequential odds ratio method and the neural network consensus model identified a similar hypothetical case of greatest risk: a Black, married man, older than 60 years, with at best a high school diploma who made between $25,000-$65,000. The work history determined by odds ratio analysis consisted of 10 years in the chemical industry with 3 yrs in the processing plant. Neural network analysis showed a similar work history with 8 years in the chemical industry and 2 years in the processing plant. DISCUSSION: Neural network outcomes are similar to sequential odds ratio calculations. This work supported previous studies by finding well known demographic risk factors for prostate cancer including certain processing jobs and chemical related jobs.

Inhibition of alpha-smooth muscle actin expression in an in vitro wound healing model by certain antibiotics.

OBJECTIVE: This study assesses the effects of antimicrobials on wound healing in an in vitro model of chicken flexor tendons in a collagen gel matrix. Two equidistant tendons were bathed in a culture medium for 28 days as fibroblasts (fb) grew from the tendon ends into the collagen gel and migrated toward each other until gap closure. Five groups of 10 paired tendons each included the control and the study groups, which received oxacillin (Ox), clindamycin (Cl), chloramphenicol (Chl), or tetracycline (Tet) in the culture medium to assess their effects on gap closure rate, fb migration, and myofibroblast alpha-smooth muscle (alpha-SM) actin expression. RESULTS: Gap closure, by day 27, was 98.5% in the controls compared with 97%, 92%, 89.5%, 21.75% in the Tet, Cl, Ox, and Chl groups. Chl retarded gap closure (p < 0.05). Fb migration was similar for all groups. In the control and Ox groups, myofibroblast expressed actin at day 5. By day 7, fb cells were clearly visible in the control, Ox, and Cl groups, whereas, only light actin was present in the Chl and Tet groups. Actin band densities for the Cl, Ox, Tet, and Chl groups were 78.4%, 62.5%, 61.7% and 26.1%, respectively, of the control group. CONCLUSION: These studies suggest that one reason certain antimicrobials impair wound healing, is due to myofibroblast inhibition of alpha-SM actin.

Neurosurgical trauma call: use of a mathematical simulation program to define manpower needs.

Resource criteria for trauma centers (TC) mandate a first plus backup neurosurgeon (NS) coverage, an unnecessary expense for TC treating few neurosurgery patients. This report uses a mathematical modeling system to define optimal NS trauma coverage. Random data from 749 patients treated with emergency neurosurgery operations (OR) within 24 hours of admission at 97 TC were used to create a 1-year profile of admission by month, day, and hour, operation times, and operation duration. These data were entered into a simulation program to define the frequency that a patient needing a NS consult would wait beyond 30 minutes because the NS was in the operating room at a trauma center with one, two, or three neurosurgeons on-call. One thousand iterations were done for each sample size of 25 to 300 patients in 25-patient increments. The probability that a patient could not be seen promptly by one NS in a trauma center operating on 25, 50, 75, or 100 patients per year is 0.23, 0.9, 1.6, and 3.66 patients per year. Fewer than one patient (0.75) per year will wait more than 30 min in a trauma center doing 225 emergency ORs when two neurosurgeons are on-call. One patient in 10 years would wait more than 30 min in a trauma center doing 300 ORs with a third NS on-call. Mathematical modeling of patient data helps define optimal hospital resources. Mandatory NS backup for TC performing fewer than 25 neurosurgery procedures is unneeded.

Thermostability of purified human pancreatic alpha-amylase is increased by the combination of Ca2+ and human serum albumin.

Pancreatic fluid from a patient with a post operative pancreatic fistula was used to isolate human alpha-amylase by means of acarbose affinity chromatography. Amylase thermostability was measured in 4 solutions: (1) EDTA-dialyzed; (2) dialyzed solution plus 0.15 mmol/l (1.0 g/dl) human serum albumin; (3) dialyzed solution plus 0.25 mmol/l (1.0 mg/dl) calcium ions; and (4) dialyzed solution with both human serum albumin and calcium ions. Amylase activity was measured at predetermined times in samples heated to 60 degrees C. Thermostability was characterized by t1/2, the time to 50% initial amylase enzyme activity. In the dialyzed solution t1/2 was 0.75 +/- 0.19 min. This rose to 1.62 +/- 0.34 min with added human serum albumin, and to 8.24 +/- 0.13 min with added calcium ions. The combination of human serum albumin and calcium ions resulted in a synergistic increase of t1/2 to 180 +/- 26 min. These findings support our contention that human serum albumin, calcium ions and possibly other body fluid constituents must be considered in any utility involving amylase thermostability as a clinically relevant diagnostic marker.

Hemodynamic, plasma volume, and prenodal skin lymph responses to varied resuscitation regimens.

The theoretical efficacy of hypertonic saline (HS) resuscitation for hemorrhagic shock purportedly stems from the osmolar extraction of intracellular fluid into the plasma. This hypothesis presumes a concomitant expansion of the interstitial fluid space. Colloid resuscitation, in theory, expands the plasma volume by extracting interstitial fluid. These hypotheses were tested in a canine-modified Wigger's model of hemorrhagic shock. Forty, male, splenectomized dogs were anesthetized and instrumented. Animals underwent a baseline equilibration period followed by shock for 120 minutes. Each animal was randomized to one of four groups and received equal amounts of Na+ either as lactated Ringer's (LR) solution, 10% dextran 40 (Dex) in normal saline, 7.5% saline (HS), or 7.5% saline plus Dex (HSD). Parameters measured at baseline, shock, and at postresuscitation 30 minutes, 60 minutes, 90 minutes, and 120 minutes, included: mean pressure (MAP), output, pulmonary capillary wedge pressure, prenodal skin lymph flow, serum and lymph albumin, wet-to-dry skin ratios, and plasma volume. MAP, cardiac output, and plasma volume were most quickly restored with LR and Dex resuscitation (MAP = 106 and 118 mm Hg) compared to HS and HSD (MAP = 98 and 92 mm Hg). Lymph flow and lymph albumin flux were best restored with LR and HSD (mean = 85 and 48 microL/min) compared to Dex and HS (mean = 36 and 37 microL/min). Wet/dry skin ratios were greatest at 60 minutes in the LR group but similar at 120 minutes in all four groups. These data suggest that interstitial fluid space remains contracted during the first hour after HS, HSD, and Dex resuscitation compared with LR resuscitation, even though the restoration of plasma volume, MAP, and cardiac output is greatest with the Dex regimen. Further studies with total body water and intracellular water are needed in this model.

Effects of interleukin-6 and its neutralizing antibodies on peritoneal adhesion formation and wound healing.

This study investigates the effects of preoperative IV administration of IL-6 and anti IL-6 on peritoneal adhesion formation and wound healing. Thirty-six male Sprague-Dawley rats (350-400 mg) were divided into three groups: control (group 1); IL-6 (group 2); and anti IL-6 (group 3). Under sterile conditions, all rats underwent a midline laparotomy. Ten cm2 of cecal serosa was abraded, the cecum further irritated with 0.1 ml of 70 per cent alcohol, and the incision closed in layers. At 3 weeks, peritoneal adhesions were graded using a score of 0 (none) to 3 (extensive, dense). Skin samples from incisional sites were examined tensiometrically (true stress and true strain), biochemically (collagen content), and histologically. Adhesion formation score was significantly increased in IL-6 group (2.78 +/- 0.44, Mean +/- SD) and decreased in anti IL-6 group (1.40 +/- 0.52) compared to control (2.00 +/- 0.50). (P < 0.03 by Kruskal Wallis test). There was no significant difference in true stress, true strain, and collagen content between the two treatment groups and controls at the 0.05 level by ANOVA. Histological analysis showed higher number of inflammatory cells and fibroblasts in IL-6 treated groups. We conclude that IL-6 plays a major role in peritoneal adhesion formation. Selective immunosuppression, using IL-6 neutralizing antibodies preoperatively, leads to a reduction of such adhesion formation without a significant effect on wound healing.

Semiautomated method for the quantitation of plasma or sera androstenedione, testosterone, and dihydrotestosterone in population studies.

A method is presented that analyzes quantitatively and reproducibly the androgens testosterone, androstenedione, and dihydrotestosterone from human sera or plasma. The chromatographic separation step generates an unattended throughput of one preparative separation per hour. Controls are built into the method to account for changing chromatographic conditions that otherwise result in shifts in retention characteristics. Separation factors for the three androgens are as follows (mean +/- standard deviation): alpha = 1.23 +/- 0.011 between androstenedione and testosterone and alpha = 1.38 +/- 0.025 between testosterone and dihydrotestosterone. Sensitivities of the method are androstenedione 5 pg, testosterone 3 pg, and dihydrotestosterone 14 pg. A study of procedural losses associated with initial sample processing, a validation, and application to two sample sets which demonstrates the methods utility for the analysis of hypoandrogenic populations (postmenopausal women) and hyperandrogenic groups (prostate cancer patients) is also reported. The precision for replicate aliquots of control plasma is androstenedione and testosterone = 5-11% CV and dihydrotestosterone = 10-20% CV.

Prediction of rib fracture injury outcome by an artificial neural network.

Outcome-based therapy is becoming the standard for assessing patient care efficacy. This study examines the ability of an artificial neural network to predict rib fracture injury outcome based on 20 intake variables determined within 1 hour of admission. The data base contained 580 patient records with four outcome variables: Length of hospital stay (LOS), ICU days, Lived, and Died. A 522-patient training set and a 58-patient test set were randomly selected. Nine networks were set up in a feed-forward, back-propagating design with each trained under different initial conditions. These networks predicted the test set outcome variables with an accuracy as high as 98% at the 80% testing level. Internal weight matrix examination indicated that age, ventilatory support, and high trauma scores were strongly associated with both ICU days and mortality. Being female, injury severity, and injury type were associated with increased LOS. Smoking and rib fracture number were low-level predictors of the four outcome variables.

An in vitro model of fibroblast activity and adhesion formation during flexor tendon healing.

We studied fibroblast activity during tendon healing with an in vitro tendon culture model. Tendons were embedded in a translucent collagen gel matrix whose porous nature permitted free nutrient diffusion, fibroblast migration out of the tendon, and microphotographic documentation of fibroblast activity. Experiments were performed using one or more tendons cultured in the same collagen gel. We identified three zones of fibroblast activity in the gel. Zone I was an area of randomly dispersed cells directly adjacent to the tendon where collagen synthesis and remodeling were probably taking place. In zone II, spindle-shaped fibroblasts were aligned pointing away from the cut tendon end forming a sunburst-like aggregate of cells. Zone II fibroblasts were responsible for formation of migration trails by exerting a mechanical force on the collagen matrix, which was evident as a local gel contraction. Zone III was the leading edge of the sunburst populated by the fastest moving fibroblasts, which responded to guidance by other cut tendon ends. We speculate that the collagen gel used in the culture system may help maintain a chemotactic concentration gradient that allows fibroblasts to locate other distal cut tendon surfaces also embedded in the collagen gel.

Analysis of protein transmembrane helical regions by a neural network.

Neural networks were used to generalize common themes found in transmembrane-spanning protein helices. Various-sized databases were used containing nonoverlapping sequences, each 25 amino acids long. Training consisted of sorting these sequences into 1 of 2 groups: transmembrane helical peptides or nontransmembrane peptides. Learning was measured using a test set 10% the size of the training set. As training set size increased from 214 sequences to 1,751 sequences, learning increased in a nonlinear manner from 75% to a high of 98%, then declined to a low of 87%. The final training database consisted of roughly equal numbers of transmembrane (928) and nontransmembrane (1,018) sequences. All transmembrane sequences were entered into the database with respect to their lipid membrane orientation: from inside the membrane to outside. Generalized transmembrane helix and nontransmembrane peptides were constructed from the maximally weighted connecting strengths of fully trained networks. Four generalized transmembrane helices were found to contain 9 consensus residues: a K-R-F triplet was found at the inside lipid interface, 2 isoleucine and 2 other phenylalanine residues were present in the helical body, and 2 tryptophan residues were found near the outside lipid interface. As a test of the training method, bacteriorhodopsin was examined to determine the position of its 7 transmembrane helices.

Correlation of high-speed tensile strength with collagen content in control and lathyritic rat skin.

Severity of lacerative skin injury depends on the applied load and the resistance of the tissue. At low (static) rates of loading there is a high degree of correlation between skin tensile strength and the degree of collagen crosslinking, with little added strength due to collagen interactions with the glycosaminoglycan matrix. We examined the effects of high (ballistic) rates of loading in order to determine the contributions to strength made by both the degree of collagen crosslinking and the collagen-matrix interaction. Tensile failure experiments were conducted using the dorsal skin of rats 1.5-6 months of age. Test specimen orientations were cut parallel and transverse to the body axis at cephalad and caudad locations on the dorsum. Tensile strength was measured at nominal strain rates of 30%/sec (low speed) and 6000%/sec (high speed) using both control and lathyrogen fed rats. Biochemical analyses were conducted to determine the amount of total and crosslinked (insoluble) collagen. In low-speed tests, there was a significant correlation (r > or = 0.900) between collagen content and skin tensile strength measured both transverse and parallel to the spine. The degree of correlation was higher with insoluble (r = 0.973) collagen content than with total (r = 0.901) collagen. The effect of a lathyrogen diet produced a significant (P < 0.001) reduction (two- to threefold) in tensile strength compared to control. In both high- and low-speed groups, tensile strength was greatest in the transverse samples with a significant correlation to collagen content (r > or = 0.858).(ABSTRACT TRUNCATED AT 250 WORDS)

Limitations of Augustinsson plots.

The Augustinsson plot transforms the Michaelis-Menten equation by graphing velocity, v, as a function of velocity/substrate, v/s. The plot is linear with a negative slope for a single class of saturable sites and curvilinear for multiple classes of sites. Statistically, Augustinsson plots suffer from the limitation that there is no true independent variable. Experimental errors form a Gaussian distribution that rests neither parallel to the v axis nor v/s axis, but rather along a line emanating from the origin. Widening the range of s values produces increasingly large errors in the calculated Km and Vm values, especially when multiple sites are present. Exact data containing no error produces accurate constants using either the Michaelis-Menten or Augustinsson equation. With Km and Vm constants previously derived by fitting data with the statistically valid Michaelis-Menten equation, the Augustinsson plot may be used accurately to display multiple site data.

Effect of interleukin-2 on biodistribution of monoclonal antibody in tumor and normal tissues in mice bearing SL-2 thymoma.

BACKGROUND: Our laboratory demonstrated previously that treatment with a tumor-specific, radiolabeled anti-Thy 1 murine monoclonal antibody (MAb) in mice can eradicate a T-cell lymphoma mass, but the doses required were toxic to normal organs. Approaches to increase MAb concentration in tumor tissue versus normal tissue may overcome this problem. Interleukin-2 (IL-2) has been shown to increase capillary permeability, as indicated by extravasation of albumin. PURPOSE: The purpose of this study was to determine whether increased extravasation of MAb at the tumor site might result in selective binding to tumor antigen, increasing localization of radiolabeled MAb at the tumor site. METHODS: We studied the effect of IL-2 on biodistribution of 1A14 MAb (anti-Thy 1.1) in normal AKR/Cum (Thy 1.2+) mice and in AKR/Cum mice bearing SL-2, a spontaneous T-cell lymphoma (Thy 1.1+), compared with biodistribution of albumin in normal mice and biodistribution of the nonreactive G3G6 MAb in tumor-bearing mice. IL-2 was given intravenously in the tail vein in doses of 0, 25,000, 50,000, 100,000, or 200,000 U twice a day for a total of seven doses over 3.5 days. Mice received injections of a mixture of 1A14 MAb (250 muCi/100 micrograms) and albumin or G3G6 MAb (145 muCi/100 micrograms) in a total volume of 200 microL at 12 hours after the last IL-2 dose. RESULTS: In normal mice, IL-2 caused a dose-dependent increase of both radiolabeled MAb and albumin in the spleen, liver, lung, and lymph node, but it spared the brain. In tumor-bearing mice, IL-2 resulted in higher levels of MAb in the tumors 72 hours after receiving injections, with 17.5% and 24.3% of the injected dose per gram of tumor present in the mice pretreated with 100,000 or 200,000 U of IL-2 twice a day for 3.5 days, compared with 13.4% in the controls. In IL-2-treated mice, levels of MAb were greater in the tumors than in critical normal organs; the differences were statistically significant for tumors versus lungs at 24 hours after injection and for tumors versus livers at 48 hours and 72 hours after injection. CONCLUSIONS: These results suggest that pretreatment with IL-2 may lead to enhanced distribution of tumor-specific MAb to the tumor site, compared with normal tissues, thus increasing therapeutic efficacy of radiolabeled MAb.

Collagen fibril formation in the presence of dexamethasone disodium phosphate.

Dexamethasone disodium phosphate was found to inhibit in vitro fibrillogenesis in a buffered collagen solution that otherwise formed in vivo like fibrils. A nonlinear relationship was observed between the steroid salt concentration and the kinetic parameter half transition time. Full fibril inhibition occurred at dexamethasone phosphate concentrations above 15 mM. At lower concentrations, sample buffers that also contained inorganic phosphate were not different from the control in activation energy of 224.3 +/- 29.3 kJ/mol (53.6 +/- 7.0 kcal/mol). The idea is advanced that the soluble steroid salt associates directly to the collagen and prevents the formation of lateral, hydrophobic interactions between adjacent collagen aggregates.

Collagen fibril formation in the presence of sodium dodecyl sulphate.

Sodium dodecyl sulphate (SDS) was used to weaken both the electrostatic and the hydrophobic interactions during collagen fibrillogenesis in vitro. The rate and extent of fibril formation as well as fibril morphology were affected by SDS concentration. Both the formation of large fibrils at 0.3 mM-SDS and the complete cessation of fibril formation at 0.5 mM-SDS were considered to be the result of SDS-induced conformational changes in the non-helical telopeptides. A possible mechanism of SDS interaction with the N-terminal and the distal region of the C-terminal telopeptides is offered.