The Ras farnesylation inhibitor BZA-5B increases the resistance to cisplatin in a human melanoma cell line.

Ras proteins have been implicated in transducing cellular responses to DNA damaging agents. We used BZA-5B, an inhibitor of Ras-farnesylation, to examine the role of Ras in cellular sensitivity to cisplatin. A human melanoma cell line (224) with a Gln61Arg mutation in N-ras was used for these studies. We report that BZA-5B treated cells show an increased resistance to cisplatin. BZA-5B treatment decreased the number of cells showing in situ DNA fragmentation and increased cell viability and clonogenic survival after cisplatin treatment. Further experiments showed that cisplatin induction of the immediate early genes c-jun and p21cip1 was not affected by BZA-5B. Finally, we show that cisplatin causes only weak activation of Jun N-terminal kinase (JNK) in a human melanoma cell line. We conclude that inhibition of Ras function decreases the sensitivity of human melanoma cells to cisplatin-induced cell death.

Side reactions in the pyroglutamic acid-7-amino-4-methylcoumarin synthesis.

N-Formylpyroglutamic acid-7-amido-4-methylcoumarine and pyroglutamyl-pyroglutamic acid-7-amido-4-methylcoumarin are the major products in the synthesis of pyroglutamic acid-7-amido-4-methylcoumarin by phosphorus pentachloride in dimethylformamide and dicyclohexylcarbodiimide under pyridine activation.

Inhibition of tumor cell growth by monoterpenes in vitro: evidence of a Ras-independent mechanism of action.

(+)-Limonene (d-limonene) and related monoterpenes show chemopreventive activity against rodent mammary carcinoma and inhibit the growth of cancer cells in vitro. One suggested mechanism for the anti-tumorigenic effect of (+)-limonene is inhibition of the post-translational isoprenylation of growth controlling Ras oncoproteins. We have here examined the growth inhibitory effect of (+)-limonene and other related monoterpenes on PANC-1 pancreas carcinoma cells (carrying a K-ras mutation) and on 12V-H-ras-transformed rat fibroblasts. (+)- and (-)-perillyl alcohol, 7-methyl-perillyl alcohol, (+)-limonene oxide and (+)-perillic acid methyl ester were all found to efficiently inhibit cell growth at 1 mM, whereas (+)-limonene caused an approximately 50% growth reduction at 5 mM. Whereas BZA-5B, an inhibitor of Ras farnesyl transferase, was found to induce morphological reversion of 12V-H-ras-transformed cells, (+)-perillyl alcohol and (+)-limonene did not induce reversion. Furthermore, monoterpenes did not decrease MAP kinase enzyme activity or collagenase promoter activity in PANC-1 cells, two functions known to be down-stream from Ras. We conclude that although effective in inhibiting the growth of tumor cells harboring activated ras oncogenes, limonene and (+)-perillyl alcohol are unlikely to act by inhibiting Ras function.

Anterior cruciate ligament reconstruction using gracilis and semitendinosus tendons. Comparison of through-the-condyle and over-the-top graft placements.

Eighty-seven patients had anterior cruciate ligament reconstruction using one-limbed gracilis and semitendinosus tendon graft. No extraarticular or associated ligamentous procedures were performed. A 44-patient subgroup was alternated on an every-other-case basis between 2 graft placements, either over the top or through the femoral condyle; 64 patients (32 of each type) returned for complete evaluations. Average followup was 2.9 years. Groups were similar in age, gender, injury chronicity, number of meniscectomies, and preoperative activity levels. No statistical differences between groups were seen in overall knee rating, range of motion, KT-1000 arthrometer measurements, isokinetic muscle testing, or one-legged hop test. Injured minus normal anterior laxity at 89 N was 2.1 +/- 2.0 mm. Isokinetic testing demonstrated an average 95% normal knee extension strength and 106% normal flexion strength, measured at 60 deg/sec. Average hop index was 95%. Eight of 64 patients evaluated demonstrated injured minus normal differences of 4.5 mm or greater. Three of 8 had frankly positive 2+ and 5 had 1+ pivot shifts; however, 4 of these returned fully to sports, and 4 returned with modifications. Overall, 55 of 64 patients returned to preinjury sports.

Measurements of light transmission through single melanophores.

A photometrical method has been developed that allows assessment of subcellular pigment migration in melanophores of the fish cockoo wrasse (Labrus ossifagus L.) The pigment migration was studied with local light spot transmission measurements. Depending on where the light beam is placed on the melanophores it is possible to study events within an area of approximately 75 microns 2. Measuring pigment translocation in different parts of a melanophore gives new possibilities to study how cell membrane receptor-mediated signals are spread within a single cell, which will increase our understanding of how receptor activating drugs exert their cellular effect. The technique can be used in pharmacological and biophysical studies and in biosensors, pharmaceutical screens, environmental detectors, etc. The method clearly has the ability to study local and small changes in light transmission due to displacement of melanophore pigment granules. Since one melanophore on the tip of an optical fibre would be enough to obtain a measurable effect, the presented technique provides the basis for future development of biosensors small enough for in vivo applications, e.g., to monitor the catecholamine levels of circulating blood.

Long-term amiodarone administration protects against global myocardial ischemia.

Reports on the effects of amiodarone on cardiac function have been variable. This study addresses the effect of long-term amiodarone administration on recovery of cardiac function after a period of global ischemia. Normotensive and spontaneously hypertensive rats were used. Normotensive rats (n = 6) received 240 mg/kg amiodarone for 4 weeks, for a total of 72 +/- 3 mg. Hypertensive rats (n = 6) received 500 mg/kg amiodarone for 4 weeks, for a total of 116 +/- 5 mg. Final myocardial concentrations of amiodarone and desethylamiodarone were 1.85 +/- 1.75 and 0.50 +/- 0.61 micrograms/g wet weight for the normotensive rats and 1.30 +/- 0.58 and 0.31 +/- 0.17 micrograms/g for the hypertensive rats (p = nonsignificant). Equal numbers of controls received sterile saline solution for 4 weeks. The hearts were excised and perfused in a Langendorff apparatus. The results indicate that, after 15 minutes of normothermic ischemia, hearts treated with this relatively low dose of amiodarone recovered a greater percentage of preischemic work (97% +/- 13%) as compared with the controls (76% +/- 17%) (p less than 0.005).

On the presence of the chromosomal proteins HMG I and HMG Y in rat organs.

Using antiserum raised against HMG I, we have shown that HMG I and HMG Y are present in perchloric acid extracts of kidney, lung, heart, brain, liver and intestine in the rat, suggesting that the expression of these proteins may not be dependent upon proliferative activity. The results also show that the ratio between HMG I and HMG Y varies between different organs.

The amino acid sequence of the chromosomal protein HMG-Y, its relation to HMG-I and possible domains for the preferential binding of the proteins to stretches of A-T base pairs.

The primary structure of the human high mobility group (HMG) protein HMG-Y has been established except for a few amino acids in the N-terminal and the C-terminal part of the protein. It was found that the sequence was identical to that of HMG-I except for a run of eleven amino acids. Like HMG-I the protein was N-terminally blocked and the palindromic sequence Pro-Arg-Gly-Arg-Pro occurred twice as in HMG-I. The binding of peptides derived from HMG-I (after thermolysin cleavage) to poly (dA-dT).poly(dA-dT) suggested that there are at least two different binding domains in the protein and that binding is not dependent upon an intact protein.