Transgenic mice expressing human ApoB95 and ApoB97. Evidence that sequences within the carboxyl-terminal portion of human apoB100 are important for the assembly of lipoprotein

The structural features of apolipoprotein (apo) B that are important for its covalent linkage to apo(a) to form lipoprotein(a) (Lp(a)) are incompletely understood. Although apoB100 cysteine 4326 is required for the disulfide linkage with apo(a), other structural features, aside from a single free cysteine residue, must be important for apoB's initial interaction with apo(a) and for facilitating the formation of the disulfide bond. To determine if sequences carboxyl-terminal to cysteine 4326 affect the efficiency of Lp(a) formation, we used "pop-in, pop-out" gene targeting in a human apoB yeast artificial chromosome to introduce nonsense mutations into exon 29 of the apoB gene. The mutant yeast artificial chromosomes, which coded for the truncated versions of human apoB, apoB95, and apoB97, were then used to express these mutant forms of apoB in transgenic mice. As judged by in vitro assays of Lp(a) formation, apoB95 (4330 amino acids) formed a small amount of Lp(a) but did so slowly. In contrast, apoB97 (4397 amino acids) formed Lp(a) rapidly, although not quite as rapidly as the full-length apoB100 (4536 amino acids). These results were supported by an analysis of double-transgenic mice expressing both human apo(a) and either apoB95 or apoB97. In mice expressing both apoB95 and apo(a), there was only a trace amount of Lp(a) in the plasma, and most of the apo(a) was free, whereas in mice expressing both apoB97 and apo(a), virtually all of the apo(a) was bound to apoB97 in the form of Lp(a). These results show that sequences carboxyl-terminal to apoB95 (amino acids 4331-4536) are not absolutely required for Lp(a) formation, but this segment of the apoB molecule, particularly residues 4331-4397, is necessary for the efficient assembly of Lp(a).     

5'-degenerate 3'-dideoxy-terminated competitors of PCR primers increase specificity of amplification

Amplification of a product in PCR with specific primers may be viewed as an artificial Darwinian-type "selection of the fittest". In other selective systems, such as general evolution, immune system and probably brain cortex, the stringency of selection is not absolute but rather degenerate, with selection of many highly fit units, not limited, however, to only the fittest. In PCR also, annealing of the primers is not absolutely specific. The subsequent amplification frequently leads to amplification of not only the desired product but also to less-specific sequences. Using theoretical analysis of the degenerate mode of selection, we predict theoretically and prove experimentally that 5'-degenerate, 3'-dideoxy-terminated competitors of PCR primers can be used to dramatically improve the specificity of PCR amplification without affecting the quantitation of the final specific product.     

Comparison of DNA-adduct and tissue-available dose levels of MeIQx in human and rodent colon following administration of a very low dose

[2-14C]2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) was administered orally (304 ng/kg body-weight dose based upon an average 70-kg-body-weight subject) to 5 human colon-cancer patients (58 to 84 years old), as well as to F344 rats and B6C3F1 mice. Colon tissue was collected from the human subjects at surgery and from the rodents 3.5 to 6 hr after administration. Colon DNA-adduct levels and tissue available doses were measured by accelerator mass spectrometry (AMS). The mean levels of MeIQx in the histologically normal colon tissue were not different among the human (97 +/- 26 pg MeIQx/g), rat (133 +/- 15 pg/g) or mouse (78 +/- 10 pg/g) tissues; and no difference existed between the levels detected in human normal and tumor tissue (101 +/- 15 pg/g). Mean DNA-adduct levels in normal human colon (26 +/- 4 adducts/10(12) nucleotides) were significantly greater (p < 0.01) than in rats (17.1 +/- 1 adduct/10(12) nucleotides) or mice (20.6 +/- 0.9 adduct/10(12) nucleotides). No difference existed in adduct levels between normal and tumor tissue in humans. These results show that MeIQx forms DNA adducts in human colon at low dose, and that the human colon may be more sensitive to the effects of MeIQx than that of mice or rats.     

Childhood cancer and parental use of tobacco: deaths from 1971 to 1976

Parental smoking data have been reabstracted from the interview records of the Oxford Survey of Childhood Cancers (deaths from 1971 to 1976). Reported smoking habits for the parents of 2587 children who died with cancer were compared with similar information for the parents of 2587 healthy controls (matched pairs analysis). Maternal daily consumption of cigarettes and paternal use of pipes or cigars were unimportant, but there was a statistically significant positive trend between paternal daily consumption of cigarettes and the risk of childhood cancer (P < 0.001). This association could not be explained by maternal smoking, social class, parental ages at the birth of the survey child, sibship position or obstetric radiography. Relations between maternal consumption of cigarettes and birth weights suggested that (maternal) smoking data were equally reliable for case and control subjects. About 14% of all childhood cancers in this series could be attributable to paternal smoking. These data were combined with smoking data from two previously published reports from the Oxford Survey (deaths from 1953 to 1955, deaths from 1977 to 1981) to obtain further information on risks for different types of cancer and different ages at onset of disease. Paternal cigarette smoking emerged as a potential risk factor both for the generality of childhood cancer and for all ages at onset.     

Early isotonic saline resuscitation from uncontrolled hemorrhage in rats

BACKGROUND: Attempts to modify traditional fluid resuscitation have been based on animal models that evaluate several variables including anesthesia. This study presents the effects of early saline resuscitation from severe uncontrolled hemorrhage unanesthetized rats. METHODS: Sixty-three female Sprague-Dawley rats were equally divided into three groups: group A, nonresuscitated; and groups B and C, resuscitated ;with isotonic saline (40 and 80 mL/kg, respectively). Hemodynamics, blood loss, survival time, and mortality were recorded for 360 minutes after the hemorrhage, which was initiated by 75% resection of the tail. RESULTS: In group C, 80 mL/kg of saline significantly lowered mortality (24% vs 76% and 71% for groups A and B, respectively) with concomitant increases in mean survival time (241 +/- 103 min vs 146 +/- 108 and 175 +/- 92 min for groups A and B, respectively). There were no statistically significant differences in blood loss, hematocrit, or hemodynamic parameters among the groups. CONCLUSIONS: Early and adequate isotonic saline resuscitation of unanesthetized rats improved outcome despite continuing hemorrhage. The significantly lower mortality rate and increased survival time were not a result of transiently improved arterial pressure and did not correlate with blood loss. No significant bleeding increases were noted in the resuscitated groups.     

Studies on compounds of cancer chemoprevention: synthesis of some amides

In search for cancer chemoprevention agents, seven new amide compounds have been synthesized. The structures have been determined based on spectral and chemical data. N-4-(ethoxycarbophenyl)-alpha-naphthamide and N-4-(ethoxycarbophenyl)-beta-naphthamide were shown to be 81% and 79% effective, respectively, for inducing different in HL-60 human promyelocytic leukemia cells at the concentration of 10(-5) mol/L in NBT tests.     

Physical activity and plasma lipoprotein lipid concentrations in men

Twenty-three apparently normal untrained men aged 20--55 participated in a 4-month self-regulated training programme ending in a marathon run. Fasting plasma and lipoprotein lipid concentrations, adipose tissue lipoprotein lipase activity, anthropometric data, alcohol consumption, smoking habits, weekly mileage run and performance on a bicycle ergometer were recorded before and after the training period. Training induced an increase in high density lipoprotein cholesterol (HDL-C) concentration which was not directly related to concomitant decreases in mean very low density lipoprotein cholesterol (VLDL-C) concentration or mean total skinfold thickness. The degree of the changes in VLDL lipids and HDL-C levels were related to pretraining values, and changes in HDL-C and VLDL triglycerides (VLDL-TG) were also associated. Initial total skinfold thickness correlated inversely with the change in VLDL-TG levels during training. The pretaining concentration of VLDL-C was related to the corresponding value for HDL-C after training. The magnitude of exercise-induced changes in VLDL-C and HDL-C levels are more related to pre-training levels than to changes in measured exercise parameters, indices of obesity or adipose tissue lipoprotein lipase activity. However, the level of adiposity of subjects at the beginning of the study influenced the response of VLDL-TG levels to increased physical activity. The data suggest that VLDL contributes to the increase in HDL-C levels with exercise but is not the major source of the increment.