Purification and chemical modification of porcine bone morphogenetic protein

Implantation of porcine bone morphogenetic protein (pBMP) in the muscle induces differentiation of mesenchymal-type cells and results in endochondral bone formation. pBMP was isolated from porcine demineralized bone matrix and purified by hydroxyapatite chromatography, Sephadex G75 gel filtration, preparative sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), preparative isoelectric focusing (IEF), and chromatofocusing fast protein liquid chromatography (FPLC). Porcine BMP has an MW of 26 K and a range of pI from 4.65 to 4.73 determined by SDS-PAGE and IEF, respectively. Reconstitution with the citrate buffer supernatant fraction enables as little as 50 micrograms of the soluble pBMP fractions to induce osteogenesis in an in vivo assay. Chemical modification studies indicate that the osteoinductive potential of the pBMP molecule depends on tyrosine, carboxyl groups, and disulfide bonds and can be increased by modification of sulfhydryl groups. Modification of arginine and tryptophan has no effect on bioactivity. By pepsin-limited proteolysis, fragments of pBMP with an MW of 6-14 K show definite, although reduced, BMP activity.     

Normothermic renal artery perfusion: A comparison of perfusates

Hypothermia and preservative perfusates have been used to decrease ischemic renal injury. This study was performed to identify the preservative function of perfusates independent of the effects of hypothermia. Rats underwent 45 minutes of renal ischemia. Rectal and renal parenchyma temperatures were monitored and maintained within 1° C of normal. Perfusates were University of Wisconsin solution (UW), Euro-Collins solution, normal saline solution, and Ringer's lactate solution. A nonperfused ischemic control and a nonischemic control group were also evaluated. Parameters evaluated included serum creatinine and blood urea nitrogen levels, renal ischemic injury grade, renal weight, and gross appearance of the injured kidney. Rats treated with UW solution were found to have a significantly lower creatinine, blood urea nitrogen, and injury grade than the other three perfused groups. The external gross appearance of the UW-treated kidneys was normal, whereas that of the other groups demonstrated moderate to severe injury. Although the mean right/left renal weight difference of the UW-treated group was lower than that of the other three groups, this was not statistically significant. Under normothermic conditions in rats, UW solution affords significant renal protection from ischemia. Euro-Collins, normal saline, and Ringer's lactate solutions display no significant protective effect.Presented at the Twentieth Annual Meeting of the Peripheral Vascular Surgery Society, New Orleans, La., June 10, 1995.     

Microvascular corrosion casting of normal tissue, transitional mucosa and adenocarcinoma in the human colon.

The microcirculatory architecture of normal tissue, transitional mucosa and adenocarcinoma of the human colon was investigated with microvascular corrosion casting (MVCC) combined with scanning electron microscopy (SEM). The study showed that the capillaries within the normal mucosa were arranged in a regular hexagonal pattern around the mucosal glands and that the microvessels of transitional mucosa mostly had lost the typical hexagonal pattern and become slightly wider in diameter. The microvessels in the tumor periphery were increased in number and disorganized, and presented large variation in morphology with claw-like formations, widened sinuses, diverticula and appendixoid patterns. Microvessels were lacking in the central areas of tumors. These morphological alterations may serve as additional indicators of tumor development.     

A comparison of arterial and venous flaps.

The survival rate and elastic properties of the anterior abdominal skin flap in Sprague-Dawley rats were studied in three groups of animals. In group 1 where the flaps were supplied by a normal artery, arterial flaps (1A) had better survival rate and elastic properties than venous flaps (1B). In group 2, where the flaps were supplied by an artery with diminished perfusion pressure, the arterial flaps (2A) still had slightly better results than venous flaps (2B). However, in group 3 where the flaps were supplied by a vein, venous flaps (3B) had better results than arterial flaps (3A).     

Effects of Nitrite Exposure on Acid–Base Balance, Respiratory Protein, and Ion Concentrations of Giant Freshwater Prawn Macrobrachium rosenbergii at Low pH

Macrobrachium rosenbergii that had been exposed individually for 24 h to 0 (control), 2, 5, 10 mg/L nitrite-N (nitrite as nitrogen) at 4.3 and 7.7 pH levels were examined for hemolymph nitrite-N, oxyhemocyanin, protein, acid–base balance, ion concentrations, and ammonia-N (ammonia as nitrogen) excretion. Hemolymph oxyhemocyanin, protein, pH, HCO₃ ⁻ , TCO₂, osmolality, and ion concentrations were inversely related to ambient nitrite-N concentration and were lower at pH 4.3. However, hemolymph nitrite-N, PO₂ and PCO₂ levels, and ammonia-N excretion were directly related to ambient nitrite-N, and were higher at pH 4.3. Ambient nitrite-N and pH level interacted to cause changes in hemolymph nitrite-N, oxyhemocyanin, protein, PO₂, and pH levels. It is concluded that for M. rosenbergii following nitrite exposure, the incorporated nitrite causes a decrease of pH and an increase of PO₂ in the hemolymph where it reduces oxyhemocyanin level; disturbs nitrogen excretion, ion regulation, and respiratory gas exchange; and may lead to a decrease of oxygen-carrying capacity, which are affected more at low pH. Received: 31 August 1996/Accepted: 9 July 1997     

Fluorescence polarization immunoassay and HPLC assays compared for measuring monoethylglycinexylidide in liver-transplant patients.

We compared fluorescence polarization immunoassay (FPIA, x) and HPLC (y) for measuring monoethylglycinexylidide (MEGX) concentrations in 119 serum samples from 61 liver-transplant donors and recipients. The correlation between the two methods was y = 1.48 micrograms/L + 0.8x (r = 0.89). The bias (mean difference) was 12 micrograms/L (0.055 mumol/L) through the MEGX concentration range measured (0-250 micrograms/L, 0-1.136 mumol/L). We observed a major difference between the two methods in samples from four recipients and one donor. Cross-reactivity in FPIA with lignocaine and two of its metabolites (glycinexylidide and 2,6-xylidine) was < 3%. Samples with high bilirubin concentrations (> 200 mumol/L) required dilution before assay of MEGX by FPIA. Although there was an increase in apparent MEGX concentrations in some samples with increased bilirubin concentrations, the relationship was not constant. Increased plasma concentrations of cholesterol and triglyceride resulted in relatively small increases in apparent MEGX concentrations.     

Different effects of intracellular and extracellular TEA on voltage-dependent K currents of acutely isolated hippocampal CA1 neurons.

Tetraethylammonium (TEA) effects on K currents were examined on either side of the membrane of hippocampal CA1 neurons by means of whole-cell voltage-clamp recording and intracellular perfusion. Recording media contained ion channel blockers to allow the selective activation of voltage-dependent K currents which consisted of a rapidly decaying component (A-current) and a delayed component. Voltage protocols were applied to separate the A-current from the delayed component. Results show that 10 mM extracellular TEA suppressed 50 +/- 11% (S.D., n = 4) of the delayed current at different levels of depolarization but had little effect on the A-current. In contrast 10 mM TEA applied by intracellular perfusion suppressed the A-current by 42 +/- 10% (S.D., n = 4) in addition to inhibiting the delayed currently 55 +/- 15% (S.D., n = 4). Both the intracellular and extracellular actions of TEA on K currents showed no voltage- nor time-dependency. The results suggest that voltage-dependent transient current (A-current) is mediated through a separate group of ionic channels distinct from those that sustained the delayed current. Furthermore, the asymmetrical effects of intracellular and extracellular TEA on the transient current are similar to those described for the A-current in molluscan neurons. This observation supports the notion that the structure of the ion channel mediating the A-current is closely conserved across different species.