Karyotype heterogeneity and unclassified chromosomal abnormalities.

In a departure from traditional gene-centric thinking with regard to cytogenetics and cytogenomics, the recently introduced genome theory calls upon a re-focusing of our attention on karyotype analyses of disease conditions. Karyotype heterogeneity has been demonstrated to be directly involved in the somatic cell evolution process which is the basis of many common and complex diseases such as cancer. To correctly use karyotype heterogeneity and apply it to monitor system instability, we need to include many seemingly unimportant non-specific chromosomal aberrations into our analysis. Traditionally, cytogenetic analysis has been focused on identifying recurrent types of abnormalities, particularly those that have been linked to specific diseases. In this perspective, drawing on the new framework of 4D-genomics, we will briefly review the importance of studying karyotype heterogeneity. We have also listed a number of overlooked chromosomal aberrations including defective mitotic figures, chromosome fragmentation as well as genome chaos. Finally, we call for the systematic discovery/characterization and classification of karyotype abnormalities in human diseases, as karyotype heterogeneity is the common factor that is essential for somatic cell evolution.

Heterogeneity of cell death.

Cell death constitutes a number of heterogeneous processes. Despite the dynamic nature of cell death, studies of cell death have primarily focused on apoptosis, and cell death has often been viewed as static events occurring in linear pathways. In this article we review cell death heterogeneity with specific focus on 4 aspects of cell death: the type of cell death; how it is induced; its mechanism(s); the results of cell death, and the implications of cell death heterogeneity for both basic and clinical research. This specifically reveals that cell death occurs in multiple overlapping forms that simultaneously occur within a population. Network and pathway heterogeneity in cell death is also discussed. Failure to integrate cell death heterogeneity within analyses can lead to inaccurate predictions of the amount of cell death that takes place in a tumor. Similarly, many molecular methods employed in cell death studies homogenize a population removing heterogeneity between individual cells and can be deceiving. Finally, and most importantly, cell death heterogeneity is linked to the formation of new genome systems through induction of aneuploidy and genome chaos (rapid genome reorganization).

Diverse system stresses: common mechanisms of chromosome fragmentation.

Chromosome fragmentation (C-Frag) is a newly identified MCD (mitotic cell death), distinct from apoptosis and MC (mitotic catastrophe). As different molecular mechanisms can induce C-Frag, we hypothesize that the general mechanism of its induction is a system response to cellular stress. A clear link between C-Frag and diverse system stresses generated from an array of molecular mechanisms is shown. Centrosome amplification, which is also linked to diverse mechanisms of stress, is shown to occur in association with C-Frag. This led to a new model showing that diverse stresses induce common, MCD. Specifically, different cellular stresses target the integral chromosomal machinery, leading to system instability and triggering of MCD by C-Frag. This model of stress-induced cell death is also applicable to other types of cell death. The current study solves the previously confusing relationship between the diverse molecular mechanisms of chromosome pulverization, suggesting that incomplete C-Frag could serve as the initial event responsible for forms of genome chaos including chromothripsis. In addition, multiple cell death types are shown to coexist with C-Frag and it is more dominant than apoptosis at lower drug concentrations. Together, this study suggests that cell death is a diverse group of highly heterogeneous events that are linked to stress-induced system instability and evolutionary potential.

Genetic and epigenetic heterogeneity in cancer: the ultimate challenge for drug therapy.

Based on the gene and pathway centric concept of cancer, current approaches to cancer drug treatment have been focused on key molecular targets specific and essential for cancer progression and drug resistance. This approach appears promising in many experimental models but unfortunately has not worked well in the vast majority of cancers in clinical settings. Many new proposals, based on the same rationale of identifying a "magic bullet" are emerging now that target the epigenetic level as well as some other new targets including metabolic regulation, genetic instability and tumor environments. In spite of the optimism resulting from these new approaches there is still a key challenge that remains regarding cancer drug therapy in the form of multiple levels of genetic and epigenetic heterogeneity. Using the recently formulated genome theory, the importance of bio-heterogeneity and its complex relationships between different levels has been discussed and in particular, the concept and methods used to monitor and target genome level heterogeneity. By briefly mentioning some newly introduced treatment options, this review further discusses the common challenges for the field as well as possible future directions of research.

Combined multicolor-FISH and immunostaining.

The combination of multicolor-FISH and immunostaining produces a powerful visual method to analyze in situ DNA-protein interactions and dynamics. Representing one of the major technical improvements of FISH technology, this method has been used extensively in the field of chromosome and genome research, as well as in clinical studies, and serves as an important tool to bridge molecular analysis and cytological description. In this short review, the development and significance of this method will be briefly summarized using a limited number of examples to illustrate the large body of literature. In addition to descriptions of technical considerations, future applications and perspectives have also been discussed focusing specifically on the areas of genome organization, gene expression and medical research. We anticipate that this versatile method will play an important role in the study of the structure and function of the dynamic genome and for the development of potential applications for medical research.

The Rhizobium leguminosarum tonB gene is required for the uptake of siderophore and haem as sources of iron.

In the N2-fixing bacterium Rhizobium leguminosarum, mutations in a homologue of tonB (tonB(Rl)) block the import of vicibactin and haem as iron sources in free-living bacteria. TonB(Rl) mutants were normal for growth with ferric dicitrate and slightly reduced for growth with haemoglobin as sole iron sources. The deduced TonB(Rl) product is larger than that of (for example) Escherichia coli, on account of an extended N-terminal domain. Transcription of tonB(Rl) was enhanced in low-Fe growth conditions; this was not controlled by Fur, nor RpoI, an Fe-regulated extracytoplasmic sigma factor. Upstream of tonB(Rl) and transcribed divergently is an operon, hmuPSTUV, whose products are homologous to ABC transporters involved in haem uptake in pathogenic bacteria. Expression of hmuPSTUV was enhanced in low-Fe conditions, and hmu mutants show slightly diminished growth on haem as sole Fe source, suggesting that there is more than one system for the uptake of this molecule. hmuPSTUV expression appears to be from three closely linked promoters. Downstream of hmuPSTUV, a gene that may encode an extracytoplasmic sigma factor was identified, but this gene, rpoZ, did not affect the transcription of tonB(Rl) or hmuPSTUV. Mutations in tonB(Rl), hmu genes and rpoZ did not affect symbiotic N(2) fixation in peas.

The purMN genes of Rhizobium leguminosarum and a superficial link with siderophore production.

We isolated a mutant of R. leguminosarum initially on the basis of reduced production of the siderophore vicibactin on chrome azurol sulfonate (CAS)/agar indicator plates. The mutation was in the purMN operon and the mutant was shown to be an adenine auxotroph and defective for nodulation of peas. The siderophore defect appears to be trivial, being due to diminished growth of the auxotroph on agar-based minimal medium, which contains unknown contaminant(s) that allow it grow poorly. Transcriptional fusions showed that purMN was transcribed at relatively high levels in media containing purines. Expression was enhanced, approximately twofold, if purines were omitted.

The immunological response of llamas (Lama glama) following experimental infection with Mycobacterium bovis.

Llamas were experimentally infected with Mycobacterium bovis in order to evaluate the axillary skin test and the ELISA as diagnostic procedures for tuberculosis in llamas (Lama glama). Six llamas were given a single intratracheal challenge with 1 of 2 doses of a recent field isolate of M. bovis and 2 llamas were left as noninfected controls. This resulted in a progressive disease in some animals with 1 mortality as early as 68 d post-infection (PI). The tuberculin skin test, at the axillary site, was positive in 4 of 5 infected llamas at 80 d PI. At 143 d PI, all 3 surviving lamas were positive, including the one which had not responded at 80 d PI. The application of skin and serological tests throughout the course of this experiment adds support for the need to further evaluate the skin test and its anamnestic effect on serodiagnosis since serological responses were generally not observed in the absence of skin testing or antibiotic treatment. The wide variation in M. bovis antigens recognized by the serological response would indicate that a diagnostic panel should include multiple antigens such as MPB70 and lipoarabinomannan (LAM). While skin testing or serology alone may be of limited value to diagnose tuberculosis in llamas, together they may offer an enhanced potential for immunodiagnosis of tuberculosis.

Tracheal intubation in ambulatory surgery patients: using remifentanil and propofol without muscle relaxants.

UNLABELLED: Using alfentanil followed by an anesthetic induction dose of propofol provides adequate conditions for tracheal intubation without neuromuscular relaxants. Remifentanil, which has a clinical onset similar to that of alfentanil, has not been investigated for this indication. Accordingly, 80 ASA physical status I and II premedicated outpatients were randomly assigned to one of four groups (n = 20/group). Remifentanil 1, 2, 3, or 4 micrograms/kg (Groups I-IV, respectively) was infused intravenously over 90 s. Sixty seconds after beginning the remifentanil infusion, propofol 2 mg/kg was infused over 5 s. Ninety seconds after the administration of propofol, laryngoscopy and tracheal intubation were attempted and graded. Clinically acceptable intubating conditions (i.e., jaw relaxed, vocal cords open, and fewer than two coughs in response to intubation) were observed in 35%, 75%, 100%, and 95% of patients in Groups I-IV, respectively. Clinically acceptable intubating conditions were significantly (P < 0.05) less likely to occur in Group I compared with all other groups. Excellent intubating conditions (i.e., vocal cords open, no movement in response to intubation) were observed in 30%, 50%, 80%, 80% of patients in Groups I-IV, respectively. Overall conditions at intubation were significantly (P < 0.05) better in Groups III and IV compared with Groups I and II. The mean time to resumption of spontaneous ventilation after induction was < 5 min in all groups. No patient manifested clinically significant muscle rigidity. The mean arterial pressure decreased 16%, 20%, 28%, 26% immediately before tracheal intubation in Groups I-IV, respectively. No patient was treated for hypotension or bradycardia. In conclusion, healthy, premedicated patients with favorable airway anatomy can be reliably intubated with good or excellent conditions 90 s after the administration of remifentanil 3-4 micrograms/kg and propofol 2 mg/kg. IMPLICATIONS: Remifentanil 3 micrograms/kg and propofol 2 mg/kg co-administered intravenously may reliably provide adequate conditions for tracheal intubation in healthy patients without neuromuscular relaxants. This combination of drugs may allow the rapid return of spontaneous ventilation.

The clinical neuromuscular pharmacology of cisatracurium versus vecuronium during outpatient anesthesia.

UNLABELLED: Neither comparisons of the clinical neuromuscular effects of cisatracurium and vecuronium nor comparative studies of their antagonism by neostigmine have been reported. In addition, the efficacy of administering cisatracurium in divided doses has not been investigated. Accordingly, we applied supramaximal electrical stimuli to the ulnar nerve of 165 ASA physical status I and II patients receiving nitrous oxide/alfentanil/propofol anesthesia. Forty-five patients received cisatracurium 5, 10, or 15 microg/kg, and the evoked response at the adductor pollicis was recorded for 15 min. One hundred-twenty patients received cisatracurium 5, 10, or 15 microg/kg or normal saline placebo followed 5 min later by either cisatracurium 100 microg/kg or vecuronium 100 microg/kg (always after placebo). Time to clinical onset (maximal ablation of single twitch response) was measured. When the evoked response spontaneously recovered to 10% of control height, neostigmine 5, 10, 30, or 50 microg/kg or placebo was administered, and recovery of neuromuscular function was recorded for the next 15 min. The clinical onset of vecuronium without priming (2.8 +/- 0.8 min) (mean +/- SD) was significantly (P < 0.05) faster than the onset of cisatracurium without priming (4.6 +/- 1.4 min). Cisatracurium 5, 10, or 15 microg/kg administered before cisatracurium 100 microg/kg significantly (P < 0.05) accelerated the time to complete ablation of the evoked response (3.9 +/- 0.9, 2.9 +/- 0.8, or 3.0 +/- 0.9 min, respectively) compared with cisatracurium 100 microg/kg without priming. The dose of neostigmine required to achieve 50% assisted recovery of the train-of-four ratio at 5 min was significantly (P < 0.05) smaller in patients who received vecuronium (29.1 [17.9-55.3] microg/kg) (mean [95% confidence interval]) compared with those who received cisatracurium (53.7 [31.6-131.5] microg/kg). Given its faster clinical onset and greater sensitivity to antagonism by neostigmine, we conclude that vecuronium may be more suitable than cisatracurium for use in outpatient anesthesia. IMPLICATIONS: We investigated the onset of muscle relaxation using intravenous vecuronium and cisatracurium and assessed the ability of neostigmine to antagonize (reverse) this effect. Our results suggest that vecuronium works faster than cisatracurium and is more sensitive to neostigmine. Vecuronium therefore may be more useful than cisatracurium in outpatient anesthesia.

Effects of valacyclovir in cats infected with feline herpesvirus 1.

OBJECTIVE: To determine whether orally administered valacyclovir can be used safely and effectively to treat cats with primary, feline herpesvirus 1 (FHV-1) infection. ANIMALS: 14 specific-pathogen-free adult cats. PROCEDURE: Cats were infected with FHV-1 strain 87-727 (300 microliters, 10(7) plaque-forming units/ml) by ocular and nasal inoculations, and were treated every 6 hours with dextrose (controls) or valacyclovir (60 mg/kg of body weight, PO). Virus shedding from both eyes and the oropharynx was monitored every 2 days by virus isolation, and subjective clinical scores were assigned daily for ocular and nasal discharge and conjunctival hyperemia. Urinalysis, CBC, and serum biochemical analysis were done prior to inoculation, and on days 2, 5, 7, 9, and 12 of infection. Differences in CBC and serum biochemical indices between groups were compared, as were differences between preinfection values and maximal postinfection values, rectal temperature, and scores for disease severity. RESULTS: All cats developed acute conjunctivitis and rhinitis typical of FHV-1 infection. Beginning between days 6 and 9, valacyclovir-treated cats became noticeably more lethargic and dehydrated than did cats of the control group. Total WBC and neutrophil counts were significantly lower in cats of the valacyclovir group. The experiment was terminated on day 12 for humane reasons. Histologic changes attributable to FHV-1 infection were similar in all cats. Additional histologic abnormalities seen only in the valacyclovir-treated cats were coagulative necrosis of the renal tubular epithelium, centrilobular atrophy and hepatic necrosis, and severe bone marrow depression. CONCLUSIONS: Cats appear to be uniquely sensitive to the toxic effects of valacyclovir, and even high doses appear not to suppress FHV-1 replication in acutely infected cats. CLINICAL RELEVANCE: Use of valacyclovir is of questionable value in cats with acute FHV-1 infection and, at high doses, the drug may be toxic.

Tracheal intubation using alfentanil and no muscle relaxant: is the choice of hypnotic important?

Administration of alfentanil followed by propofol intravenously (IV) without neuromuscular blockade for induction of anesthesia provides adequate conditions for tracheal intubation. Other hypnotic drugs have not been thoroughly investigated in this regard. Accordingly, 140 ASA physical status I and II premedicated outpatients were randomly assigned to one of seven groups (n = 20/group). Patients in Groups I-VI received alfentanil 40 microg/kg followed by etomidate 0.3 mg/kg, propofol 2 mg/kg, or thiopental 4 mg/kg. One half of these patients (Groups II, IV, VI) also received lidocaine 1 mg/kg IV prior to the administration of the above drugs. Patients in group VII received d-tubocurarine 3 mg followed by thiopental 4 mg/kg and succinylcholine 1 mg/kg. Ninety seconds after induction, laryngoscopy and endotracheal intubation were attempted and graded. Patients in Group V (alfentanil/thiopental) were significantly (P < 0.05) more likely to have a clinically unacceptable response to intubation (55%) (e.g., vigorous coughing, purposeful movement, or requirement for succinylcholine to complete intubation) compared with patients who received propofol (35%) or etomidate (20%). Alfentanil/etomidate yielded intubation conditions comparable to those achieved with alfentanil/propofol and d-tubocurarine/thiopental/succinylcholine. Lidocaine appeared to improve intubating conditions, although this improvement did not reach statistical significance. The results suggest that healthy, premedicated patients with favorable airway anatomy who have received alfentanil 40 microg/kg can be reliably tracheally intubated 90 s after administration of propofol 2 mg/kg or etomidate 0.3 mg/kg.

Serum chemistry and hematology values for anesthetized American bullfrogs (Rana catesbeiana).

Samples taken from seven male and seven female adult American bullfrogs (Rana catesbeiana) were evaluated by complete blood count and serum chemistry to establish baseline data on commercially available frogs destined for laboratory use. Differences between sexes were analyzed and females had higher plasma protein, calcium, and sodium levels.

The haemodynamic effects of rocuronium and vecuronium are different under balanced anaesthesia.

BACKGROUND: Rocuronium has been reported to have minimal haemodynamic effects. However, this conclusion has been drawn primarily from investigations conducted under narcotic-based anaesthesia. This study was designed to evaluate the cardiovascular effects of rocuronium under isoflurane/N2O/fentanyl anaesthesia and to compare rocuronium's haemodynamic effects to those of vecuronium and pancuronium. METHODS: Anaesthesia was induced with fentanyl 2 micrograms/kg, thiopentone 4 mg/kg, and suxamethonium 0.5 mg/kg in 75 ASA I or II patients. After tracheal intubation, anaesthesia was maintained with isoflurane 0.5% and N2O 50% in oxygen. Five min after intubation (baseline), patients randomly received either vecuronium 100 micrograms/kg, rocuronium 600 micrograms/kg, rocuronium 900 micrograms/kg, rocuronium 1200 micrograms/kg, or pancuronium 140 micrograms/kg. One min after administration of muscle relaxant, mean arterial pressure (MAP) and heart rate (HR) were recorded and were subsequently measured at 1-min intervals for the next 4 min. RESULTS: HR decreased significantly (P < 0.05) at all times compared to baseline in patients receiving vecuronium. HR significantly (P < 0.05) increased in those receiving rocuronium 1200 micrograms/kg or pancuronium. Patients who received vecuronium had a significant (P < 0.05) decrease in MAP at all times compared to baseline. Comparing results between groups, patients who received rocuronium or pancuronium had significantly (P < 0.05) higher MAP compared to those administered vecuronium. CONCLUSION: The haemodynamic effects of rocuronium and vecuronium are different under balanced anaesthesia. Rocuronium may attenuate the fall in MAP that often occurs under balanced anaesthesia without surgical stimulation.

Strong ion difference approach to acid-base imbalances with clinical applications to dogs and cats.

Stewart's strong ion difference, first introduced more than 10 years ago, offers an innovative approach for the analysis of non-respiratory acid-base disorders. This article addresses the concept of strong ion difference and discusses its clinical applications. A brief review of base excess and anion gap is also included. Clinical cases are provided to demonstrate a step-by-step method using strong ion difference to evaluate nonrespiratory acid-base imbalances.

A mixture of mivacurium and rocuronium is comparable in clinical onset to succinylcholine.

STUDY OBJECTIVES: To compare the clinical onset and duration of a combination of mivacurium and rocuronium with succinylcholine, and to determine the efficacy of this mixture for rapid tracheal intubation. DESIGN: Observer-blind prospective study. SETTING: Teaching hospital. PATIENTS: 70 ASA status I and II patients having general anesthesia for elective surgery. MEASUREMENTS AND MAIN RESULTS: After induction of general anesthesia, patients randomly received succinylcholine 1.0 mg/kg, rocuronium 0.6 mg/kg, or a combination of rocuronium 0.6 mg/kg and mivacurium 0.15 mg/kg. Evoked muscular response at the adductor pollicis was measured by mechanomyography. The time from injection of muscle relaxant(s) to ablation of T1 (clinical onset) and recovery of T1 to 25% of control height (clinical duration) was recorded. Intubating conditions 45 seconds after administration of muscle relaxants were assessed. There was no significant difference in clinical onset time between succinylcholine (mean +/- SD, 47.4 +/- 6.5 seconds) and the combination of mivacurium-rocuronium (51.2 +/- 13.4 seconds). Intubating conditions with mivacurium-rocuronium were comparable to those of succinylcholine. The clinical duration of rocuronium 0.6 mg/kg (38.9 +/- 12.3 minutes) was prolonged by the addition of mivacurium (49.0 +/- 9.6 minutes). CONCLUSIONS: This combination of mivacurium and rocuronium is comparable to succinylcholine in both clinical onset time and quality of intubating conditions. When rapid onset of dense neuromuscular blockade and intermediate clinical duration is desirable, this mixture may be an acceptable alternative to succinylcholine.

Nebulized tetracaine attenuates the hemodynamic response to tracheal intubation.

BACKGROUND: Local anesthetics, principally lidocaine, are commonly administered in an attempt to blunt the hemodynamic response to tracheal intubation. There are no prior investigations evaluating the use of tetracaine in this setting. METHODS: In the present study, 30 female patients (ASA I-II) scheduled for elective surgery were randomized to receive inhaled nebulized tetracaine 0.5 mg/kg or nebulized saline. Subsequently, anesthesia was induced with thiopental 5mg/kg and succinylcholine 1.0 mg/kg. Sixty seconds later, the trachea was intubated. Vital signs were recorded at intubation and at 1-minute intervals for the next 5 minutes. RESULTS: Patients treated with tetracaine had a significantly (P < 0.05) lower mean heart rate at intubation and all subsequent times. Similarly, there were significant differences (P < 0.05) in mean arterial pressure between tetracaine and control patients at all times post-intubation except at 2 minutes. The inhalation of nebulized tetracaine produced no adverse side effects. CONCLUSION: We conclude that nebulized tetracaine significantly attenuates the hemodynamic response to tracheal intubation.

Hematologic, blood gas, blood chemistry and serum mineral values for a sample of clinically healthy adult goats.

Fifty-five hematologic, blood gas, blood chemistry, and serum mineral values were determined from a random sample of 29 clinically healthy goats from an equal number of randomly selected herds in the piedmont of North Carolina. Descriptive statistics and equations for calculated values are presented. The internal and external validity of the data is discussed.

Clinical and laboratory findings in chronic conjunctivitis in cats: 91 cases (1983-1991).

Clinical findings and laboratory test results from 91 cats with chronic conjunctivitis were studied to determine the causes of the disease and the sensitivity of diagnostic procedures used, and to identify correlations between results of various diagnostic procedures and clinical or signalment variations. Mean age of affected cats was 2.9 +/- 2.7 years (+/- SD), with a range from 1 month to 11 years. Conjunctivitis was more likely to be bilateral (56 cats) than unilateral (35 cats). In cats tested for FeLV or feline immunodeficiency virus infection, 15 and 8.5%, respectively, of the results were positive, compared with 4 and 2.6% for the general hospital population. Culturing or immunofluorescent assay (IFA) for feline herpesvirus 1 (FHV-1) and Chlamydia psittaci IFA resulted in identification of pathogens (positive test results) in 19% (FHV-1) and 18% (C psittaci) of tested cats. For FHV-1, culturing was more sensitive than was IFA, yielding positive results in 19 vs 8.8% of cases. In only 1 cat were FHV-1 and chlamydiae recovered. The probability of positive test results for FHV-1 or chlamydiae was unrelated to concurrent corneal disease, unilateral vs bilateral involvement, or age. Cause of conjunctivitis could not be definitively determined in the remaining 35 cases tested for both agents. Bacterial species considered to be potentially pathogenic were isolated from conjunctival sac specimens in only 1 of 38 attempts. Cytologic changes considered compatible with chlamydial or FHV-1 infection (intracytoplasmic inclusions or multinucleated epithelial cells, respectively) were found in 8 and 5 cases, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Episodic weakness associated with exertional lactic acidosis and myopathy in Old English sheepdog littermates.

Two Old English Sheepdog littermates were evaluated for weakness that developed during periods of minimally intense exercise. Lactic acidosis accompanied by increased muscle enzyme activity, an increased lactate/pyruvate ratio, and increased venous PO2 supported the possibility of defective mitochondrial oxygen use. Electromyographic abnormalities included increased insertional activity and complex repetitive discharges. Muscle alterations included scattered myofiber necrosis, abundant endomysial connective tissue, excessive glycogen accumulation, and greater than normal numbers and vacuolation of mitochondria. A distinctive pattern of subsarcolemmal mitochondrial aggregates, referred to as "ragged red fibers" in human mitochondrial myopathies, was observed in muscle biopsy samples from 1 dog. Several features of the disease in these dogs, including onset of weakness during early life, simultaneous disease in littermates, subtle nonprogressive weakness of at least 3 years' duration, and partial reversibility of lactic acidosis following rest were suggestive of an inborn error of metabolism, consistent with mitochondrial myopathy.