Aggressive subtypes of human colorectal tumors frequently exhibit amplification of the c-myc gene

Expression of the c-myc gene is often elevated in human colorectal tumors, but reported amplification of the locus is rare. Here we demonstrate that modest amplification of c-myc is frequently found in aggressive subtypes of colorectal cancer. Careful quantitation of c-myc copy number has shown amplification in 53.8% (7/13) mucinous tumors, 42.3% (3/7) poorly differentiated tumors and a single poorly differentiated APUD tumor. This contrasts with amplification in 6.9% (2/29) moderately to well differentiated tumors, a value which is in agreement with that in previously published reports. Such changes in gene copy number may represent an important aspect of the genomic alterations which accumulate during the development of colorectal tumors.     

Comparative Pharmacodynamics of Pancuronium,Cisatracurium, and CW002 in Rabbits

Pancuronium is a long-duration neuromuscular blocking drug (NMBD) that has been used in anesthetized rabbits at 0.1 mg/kg. However, there are limited data regarding the time course for recovery from this dose either spontaneously or with pharmacologic reversal. Here we defined the potency, onset, and recovery characteristics for the intermediate-duration NMBD cisatracurium and CW002 (a novel cysteine-inactivated molecule) in the rabbit, and test the hypothesis that these drugs may be alternatives to 0.1 mg/kg pancuronium for survival procedures. New Zealand white rabbits anesthetized with isoflurane were studied in a cross-over design. Potencies of cisatracurium and CW002 were defined as the effective dose for 95% depression of evoked muscle twitch (ED₉₅). Responses to 3×ED₉₅ were used to define onset (time to maximal effect), recovery index (RI; time from 25% to 75% recovery of twitch), and duration (time to complete recovery). Responses to all drugs were determined with and without reversal by neostigmine–glycopyrrolate or l-cysteine. CW002 was 4-fold more potent than was cisatracurium, but their onset, RI, and duration were similar. Pancuronium had similar onset and RI but longer duration, compared with cisatracurium and CW002. Reversal shortened the recovery index and duration for all 3 drugs. At 3×ED₉₅, cisatracurium and CW002 had the same onset as did standard-dose pancuronium, but durations were shorter and more predictable. In addition, CW002 can be reversed without the potential side effects of cholinergic manipulation. We conclude that cisatracurium and CW002 are viable alternatives to pancuronium for survival studies in rabbits.Abbreviations: ED, effective dose; NMBD, neuromuscular blocking drug; RI, recovery indexNeuromuscular blocking drugs (NMBD) are frequently administered during surgery to provide profound muscle relaxation that cannot be achieved with general anesthesia alone. Although new NMBD have been introduced into human clinical practice over the last 20 y, surgical protocols involving many animal species continue to use older NMBD, largely because of tradition and a lack of knowledge concerning the potency and pharmacodynamics of new agents. Optimal pharmacologic management during and after experimental surgery in animal models is particularly important for both animal welfare and study outcomes.Pancuronium bromide, a long-acting, nondepolarizing NMBD first synthesized in 1964,⁴ continues to be used in laboratory animals including rabbits.^20,40,42 Beginning in the 1980s, a pancuronium dose of 0.1 mg/kg was reported for rabbits and has persisted over time.^{12,13,16,18,30,42} However, there are limited data defining the time course for complete recovery from this dose, either spontaneously or after pharmacologic reversal. In human medicine, intermediate-duration nondepolarizing neuromuscular blockers such as the benzylisoquinolinium compound cisatracurium besylate are now preferred, largely due to the potential for residual postoperative muscle weakness after pancuronium.^7,37 To date, the relative potency, onset time, duration, and reversal characteristics for cisatracurium in rabbits remain unclear.Over the last 10 y, a new class of neuromuscular blockers has been developed that is inactivated by interaction with the endogenous amino acid l-cysteine. The first in this series was gantacurium, an asymmetric benzylisoquinolinium α-chlorofumarate with an ultrashort duration due to very rapid molecular inactivation by endogenous l-cysteine.^6,25,33 More recently, CW002, a nonhalogenated, symmetrical, benzylisoquinolinium fumarate, was developed to interact more slowly with l-cysteine,^15,25 and provide an intermediate duration (approximately 40 min). Importantly, research has now shown that CW002 can be reversed at any time by injection of exogenous l-cysteine, a process that has been termed ‘facilitated molecular inactivation.’³⁸ Functionally, cysteine binds to a portion of the CW002 molecule to produce a conformational change that dramatically decreases affinity for nicotinic receptors at the motor endplate.³³ This modification is irreversible, and the CW002–cysteine adduction product is metabolized. From the clinical perspective, the ability to facilitate molecular inactivation of CW002 by injecting cysteine, an endogenously produced substance with no capacity for hypersensitivity reactions,¹⁵ at any time allows clinicians to tailor the duration of muscle relaxation to clinical need: a single dose can last 4 min or 40 min. In addition, unlike with most conventional NMBD, even complete paralysis due to CW002 can be reversed rapidly, and this process does not require the use of conventional anticholinesterase or antimuscarinic drugs, thus avoiding their potential side effects.^24,25,33 To date, the potency and duration of CW002 in rabbits are unknown. We hypothesized that both cisatracurium and CW002 would have recovery profiles that are preferable to ‘standard-dose’ (0.1 mg/kg) pancuronium, rendering them viable alternatives for use in rabbits undergoing survival procedures. To test this hypothesis, we first defined the potencies of cisatracurium and CW002 in rabbits. Subsequently, the spontaneous and reversed recovery profiles for each drug given at clinically relevant doses were compared with those for the standard pancuronium dose.     

Increased expression of the G gamma and A gamma globin genes associated with a mutation in the A gamma enhancer

We have previously described a unique type of delta beta-thalassemia in a Chinese family characterized by increased expression of the G gamma and A gamma fetal globin genes in the absence of a large deletion in the beta-globlin gene cluster. Our earlier study of the beta-globin gene on this delta beta-thalassemia chromosome showed a promoter mutation in the TATA box. In this report, we describe the results of our study of the fetal globin domain of this delta beta-thalassemia chromosome. We have cloned a 13-kb DNA fragment that includes the G gamma and the A gamma genes and the 3' A gamma enhancer element of this delta beta-thalassemia chromosome. DNA sequence analysis of the G gamma and A gamma-globin genes including their promoters did not show any mutations, but analysis of the putative enhancer element downstream from the A gamma-globin gene showed a C to T substitution 2,401 nucleotides downstream from the A gamma cap site. We performed DNA linkage analysis to determine if this mutation is unique to this chromosome or represents a common polymorphism. Our linkage analysis showed that this mutation is not a common polymorphism and that it is also not an intrinsic part of the haplotype of the chromosome on which it was found. We also studied the interaction of nuclear proteins from erythroid and nonerythroid cells with the DNA sequences surrounding this mutation. We have shown by in vitro DNase I footprinting that this mutation falls within a region that is occupied by a novel DNA-binding protein that binds to this site in nuclear extracts from erythroid, but not nonerythroid cells. The binding of this nuclear protein to DNA appears to be dependent on GATA-1 binding to an adjacent GATA-1 site. We have also developed a new functional assay to compare the activity of the normal and mutant A gamma enhancer elements in erythroid cells. Analysis of the activity of the mutant enhancer shows that the mutation completely eliminates all enhancer activity in this assay. These findings suggest that this mutation of the A gamma enhancer on a chromosome that carries a partially inactivated beta-globin gene may be responsible for the increased expression of both gamma-globin genes seen in this condition.     

Poikilocytic hereditary elliptocytosis associated with spectrin Alexandria: an alpha I/50b Kd variant that is caused by a single amino acid deletion

Hereditary elliptocytosis (HE) is a heterogeneous disorder of red blood cells frequently associated with abnormal limited tryptic digestion of the alpha I domain of spectrin and impaired spectrin dimer self- association. We studied two related individuals with poikilocytic hereditary elliptocytosis (HE) of different severity. Limited tryptic digestion of spectrin from these individuals showed the presence of a variant alpha I/50b Kd peptide at the expense of the normal alpha I/80 Kd peptide. Amino acid sequence analysis of the abnormal peptide showed that the proteolytic cleavage occurred after the arginine at position 470 of the alpha spectrin chain. Spectrin from these patients had an impaired ability to undergo self-association, as evidenced by increased amounts of spectrin dimers in 4 degrees C extracts of erythrocyte membrane from affected individuals. The polymerase chain reaction was used to study the DNA sequence of the alpha spectrin gene encoding the region of the alpha spectrin chain surrounding the abnormal proteolytic cleavage site. We detected the in-frame deletion of the trinucleotide CAT, encoding histidine 469, two amino acid residues to the N-terminal side of the abnormal proteolytic cleavage site between residues 470 and 471. Similar to many other defects of spectrin associated with HE, this deletion occurs in helix three of repeat 5 of the proposed triple helical model of spectrin repeats.     

INDIVIDUALISATION OF HIV THERAPY: THE CLINICIAN'S PERSPECTIVE

SUMMARY Assessment of clinical and laboratory markers of HIV infection may be used to individualise antiretroviral therapy. Data suggest that measures of viral load may be of considerable value as both a baseline and dynamic therapy marker, making these tools particularly useful in driving therapeutic decisions. Similarly, in-vitro data regarding intracellular pharmacokinetics and activity, resistance patterns and potential synergy of antiretroviral agents may be used to guide selection of optimal treatment regimens in clinical practice.