Origin recognition complex binding to a metazoan replication origin.

The initiation of DNA replication in eukaryotic cells at the onset of S phase requires the origin recognition complex (ORC) [1]. This six-subunit complex, first isolated in Saccharomyces cerevisiae [2], is evolutionarily conserved [1]. ORC participates in the formation of the prereplicative complex [3], which is necessary to establish replication competence. The ORC-DNA interaction is well established for autonomously replicating sequence (ARS) elements in yeast in which the ARS consensus sequence [4] (ACS) constitutes part of the ORC binding site [2, 5]. Little is known about the ORC-DNA interaction in metazoa. For the Drosophila chorion locus, it has been suggested that ORC binding is dispersed [6]. We have analyzed the amplification origin (ori) II/9A of the fly, Sciara coprophila. We identified a distinct 80-base pair (bp) ORC binding site and mapped the replication start site located adjacent to it. The binding of ORC to this 80-bp core region is ATP dependent and is necessary to establish further interaction with an additional 65-bp of DNA. This is the first time that both the ORC binding site and the replication start site have been identified in a metazoan amplification origin. Thus, our findings extend the paradigm from yeast ARS1 to multicellular eukaryotes, implicating ORC as a determinant of the position of replication initiation.

Maintenance of the DNA puff expanded state is independent of active replication and transcription.

The maintenance of the expanded state of DNA puffs II/2B and II/9A in polytene chromosomes from stage 14 x 7 Sciara coprophila salivary glands was assayed after inhibition of RNA synthesis, DNA synthesis, or both processes together. Heat shock conditions were established in order to inhibit transcription. Polypeptides of Mr 72,000 and 36,000 were produced in Sciara after heat shock. The gene encoding the Mr 72,000 polypeptide, the homolog of Drosophila hsp70, was cloned. In situ hybridization detected Sciara hsp70 at bands 4A and 17C on chromosome IV. Sciara hsp70 encodes a 2.3 kb heat shock mRNA. DNA puffs (e.g., DNA puffs 2B and 9A on chromosome II) remained fully expanded even after inhibition of transcription by heat shock or actinomycin D, or after inhibition of DNA replication by aphidicolin, or inhibition of both RNA synthesis and DNA synthesis together by actinomycin D plus aphidicolin. Therefore, maintenance of the DNA puff expanded state in Sciara does not require ongoing transcription and/or replication. Mechanisms for initiation and for maintenance of puffs (open chromatin structure) are discussed.

Tizanidine in the management of spasticity and musculoskeletal complaints in the palliative care population.

Spasticity and other muscle symptoms in the palliative care patient can contribute to suffering, significantly detracting from overall quality of life. Current therapy primarily includes use of centrally acting muscle relaxants, which are beneficial in treating some symptoms, but frequently have extensive side effects, such as sedation and muscle weakness. Tizanidine, a central alpha 2 adrenergic agonist, has been shown in clinical studies to be as effective as other commonly used antispastic agents, but without debilitating muscle weakness. Tizanidine can cause sedation, which is minimized by dose titration. When taken at night, patients report improvement in getting to sleep and little drowsiness or "hangover sensation" upon waking. Tizanidine is potentially helpful to many palliative care patients with chronic muscle pain and sleep disturbances.

The prognostic value of proliferation indices: a study with in vivo bromodeoxyuridine and Ki-67.

Proliferation indices are intended to help patients and clinicians make treatment decisions. We have previously demonstrated that a proliferation index based on in vivo labeling of S-phase cells with bromodeoxyuridine (BrdUrd) correlates with Ki-67 labeling index (LI). We now compare the prognostic value of these indices. With written consent, we gave 129 women with biopsy confirmed breast cancer 200 mg/M2 BrdUrd during 30 min immediately preceding surgery. We used IU-4 anti BrdUrd antibody to count the immunohistochemical labeling index (LI) of DNA-incorporated BrdUrd in 2,000 cells and MIB-1 to count Ki-67 (118 cases). Patients received standard surgical and adjuvant treatment. No patients were lost to follow-up and patients were followed a minimum of 2 (median 5.1) years. We compared survival and recurrence in tumors with high vs low labeling indices. We found that women in the low BrdUrd LI group had better disease free survival (92% vs 67% 5-yr DFS p = 0.001) and overall survival (94% vs 70% 5-yr OS, p = 0.0001) than those with a high LI. In comparison, a low Ki-67 index predicted better OS (87% vs 80% 5-yr OS, p = 0.020) and a trend for better DFS (84% vs 72% DFS p = 0.055). The apparent superiority of BrdUrd LI over Ki-67 LI is likely due to chance (p = 0.18). In multivariate survival analyses we found that BrdUrd LI proliferative index significantly improves prediction of DFS or OS even when node status, age or tumor size is in the model. We conclude that markers of proliferation are useful adjuncts in predicting patient prognosis.

Use of continuous ambulatory infusions of concentrated subcutaneous (s.q.) hydromorphone versus intravenous (i.v.) morphine: cost implications for palliative care.

Health care practitioners are increasingly under pressure to curtail spending while trying to deliver excellent patient care. These issues are also affecting palliative care, particularly now that palliative care programs are expanding. A comparison of cost-effectiveness and feasibility of using continuous subcutaneous (s.q.) ambulatory infusion of hydromorphone versus intravenous (i.v.) ambulatory morphine is illustrated in this study. With the high doses of morphine required in chronic cancer pain, the use of subcutaneous morphine is not feasible due to the volume of solution required to be delivered. Hydromorphone can be prepared in concentrated solutions enabling it to be delivered by the subcutaneous route. Morphine stability data are available. However, hydromorphone stability has only been verified for seven days; thus, stability data were needed post-seven days. Concentrations of 10 mg/ml, 20 mg/ml, 50 mg/ml, and 100 mg/ml, in 0.9 percent normal saline or dextrose 5 percent water, were analyzed via high-performance liquid chromatography (HPLC) at seven and 28 days. Cost comparisons of supplies and associated costs with subcutaneous versus intravenous solutions were obtained. Hydromorphone was found to be stable for 28 days in both dilutants. Cost analysis of a hydromorphone 28-day supply resulted in substantial savings over the equivalent costs of morphine infusions.

Normal radiographic values for cartilage thickness and physeal angle in the pediatric hip.

Ninety-three standing anteroposterior (AP) pelvis roentgenograms in 87 patients were measured for a total of 186 normal hips in children aged 1-17 years. For each hip, the physeal angle relative to the floor, the physeal angle relative to the pelvis, the cartilage thickness perpendicular to the floor, and the cartilage thickness perpendicular to the physis were measured and recorded. The physeal angle varied from ages 1-7 years, stabilizing at age 8 at a mean of 23 degrees . Physeal angle is best measured relative to the floor because pelvic obliquity introduces significant variability to the measurements. Cartilage thickness ("joint space") declined after age 7 years, with measurements in three statistically distinct groups. There was a statistically significant difference between cartilage-thickness measurements of boys versus girls, with girls showing a slightly smaller cartilage thickness than boys. Cartilage thickness measured perpendicular to the floor was not statistically significantly different from that measured perpendicular to the physis. We describe and recommend standard measurement techniques for physeal angle and cartilage thickness. These established normal values may be helpful in the diagnosis and evaluation of coxa vara and chondrolysis, and in identifying the head at risk for slipped capital femoral epiphysis or Legg-Calvé-Perthes disease.

Characterization of the internalization pathways for the cystic fibrosis transmembrane conductance regulator.

Mutations in the gene encoding the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) chloride channel give rise to the most common lethal genetic disease of Caucasian populations, CF. Although the function of CFTR is primarily related to the regulation of apical membrane chloride permeability, biochemical, immunocytochemical, and functional studies indicate that CFTR is also present in endosomal and trans Golgi compartments. The molecular pathways by which CFTR is internalized into intracellular compartments are not fully understood. To define the pathways for CFTR internalization, we investigated the association of CFTR with two specialized domains of the plasma membrane, clathrin-coated pits and caveolae. Internalization of CFTR was monitored after cell surface biotinylation and quantitation of cell surface CFTR levels after elution of cell lysates from a monomeric avidin column. Cell surface levels of CFTR were determined after disruption of caveolae or clathrin-coated vesicle formation. Biochemical assays revealed that disrupting the formation of clathrin-coated vesicles inhibited the internalization of CFTR from the plasma membrane, resulting in a threefold increase in the steady-state levels of cell surface CFTR. In contrast, the levels of cell surface CFTR after disruption of caveolae were not different from those in control cells. In addition, although our studies show the presence of caveolin at the apical membrane domain of human airway epithelial cells, we were unable to detect CFTR in purified caveolae. These results suggest that CFTR is constitutively internalized from the apical plasma membrane via clathrin-coated pits and that CFTR is excluded from caveolae.

Fatal nefopam overdose.

Nefopam is a non-opioid analgesic agent with a central mode of action involving activation of descending pain-modulating pathways and inhibition of synaptosomal uptake of hydroxytryptamine, norepinephrine and dopamine. Adverse effects during therapeutic use and after overdose of nefopam are known to involve the central nervous system (confusion and convulsions), the cardiovascular system (tachycardia and palpitations) and the kidneys (oliguria and renal failure). We report a death after nefopam overdose in a young woman who exhibited many of these features. It is only the second case of death after nefopam overdose in the literature.

A randomized double-blind study of the effect of distant healing in a population with advanced AIDS. Report of a small scale study.

Various forms of distant healing (DH), including prayer and "psychic healing," are widely practiced, but insufficient formal research has been done to indicate whether such efforts actually affect health. We report on a double-blind randomized trial of DH in 40 patients with advanced AIDS. Subjects were pair-matched for age, CD4+ count, and number of AIDS-defining illnesses and randomly selected to either 10 weeks of DH treatment or a control group. DH treatment was performed by self-identified healers representing many different healing and spiritual traditions. Healers were located throughout the United States during the study, and subjects and healers never met. Subjects were assessed by psychometric testing and blood draw at enrollment and followed for 6 months. At 6 months, a blind medical chart review found that treatment subjects acquired significantly fewer new AIDS-defining illnesses (0.1 versus 0.6 per patient, P = 0.04), had lower illness severity (severity score 0.8 versus 2.65, P = 0.03), and required significantly fewer doctor visits (9.2 versus 13.0, P = 0.01), fewer hospitalizations (0.15 versus 0.6, P = 0.04), and fewer days of hospitalization (0.5 versus 3.4, P = 0.04). Treated subjects also showed significantly improved mood compared with controls (Profile of Mood States score -26 versus 14, P = 0.02). There were no significant differences in CD4+ counts. These data support the possibility of a DH effect in AIDS and suggest the value of further research.

The functional relationship between in vivo bromodeoxyuridine labeling index and Ki-67 proliferation index in human breast cancer.

Proliferation indices are used, along with other parameters, to estimate the risk of recurrence of breast cancer for individual patients. Because it is unlikely one index will be practical for all patients, it is important to understand the relationship between various indices of proliferation. For this reason, we compared a proliferation index based on in vivo labeling of S-phase tumor cells with the thymidine analog bromodeoxyuridine (BrdUrd), to a proliferation index based on an estimate of the growth fraction with the MIB-1 antibody to the Ki-67 antigen. With informed consent, we gave 145 patients 200 mg/m2 BrdUrd intravenously just prior to surgical removal of breast cancer. On histology sections, we visually counted S-phase cells which had incorporated BrdUrd using the Br-3 antibody which is specific to DNA-incorporated BrdUrd, and we counted cells in the growth fraction using the MIB-1 antibody to the Ki-67 antigen. We found that both indices were positively correlated with tumor size, number of positive nodes, and tumor grade, and both were negatively correlated with age and estrogen-progesterone receptor positivity. Using a linear functional relationship model, we found that the best (i.e. the maximal) fit between the two indices (correlation coefficient 0.79; p < 0.0001) occurred when each index was square root transformed, as is appropriate when counts follow a Poisson distribution. When we used the median as a cutpoint for each index, the classification of 19 percent of data pairs changed depending upon which index was used. We also estimated that the Ki-67 intercept (1.02 +/- 0.25) was significantly greater than zero. We conclude that the BrdUrd index of DNA synthesis in S-phase correlates highly with the MIB-1 index of the growth fraction, and both indices correlate well with other parameters of tumor aggressiveness. Because this correlation is driven by concordance of the extremes of high and low counts, clinical comparison will be necessary to determine which is the better prognostic marker for human breast cancer.

The human homologue for the Caenorhabditis elegans cul-4 gene is amplified and overexpressed in primary breast cancers.

Amplification is a key mechanism whereby a cancer cell increases the message level of genes that confer a selective advantage when they are overexpressed. In breast cancer, there are many chromosome regions present in multiple copies relative to overall DNA copy number (amplicons), and their target genes are unknown. Using differential display, we have cloned and sequenced the full coding region of a candidate amplicon target gene located on chromosome 13. This candidate is the human homologue of the Caenorhabditis elegans cul-4 gene, cul-4A, a member of the novel cullin gene family, which is involved in cell cycle control of C. elegans. cul-4A was amplified and overexpressed in 3 of 14 breast cancer cell lines analyzed, and it was overexpressed in 8 additional cell lines in which it was not amplified. The latter observation, indicating that its overexpression can occur by mechanisms other than gene amplification, suggests that cul-4A plays a key role in carcinogenesis. Moreover, cul-4A was found to be amplified in 17 of 105 (16%) cases of untreated primary breast cancers, and 14 of 30 cases analyzed (47%) were shown by RNA in situ hybridization to overexpress cul-4A. These results suggest that up-regulation of cul-4A may play an important role in tumor progression.

Antecedent antimicrobial use increases the risk of uncomplicated cystitis in young women.

To examine whether antecedent antimicrobial use influenced subsequent relative risk of urinary tract infection (UTI) in premenopausal women, data were analyzed from two cohorts of women observed prospectively for 6 months to determine risk factors for UTI. Using a Cox proportional hazards model to adjust for covariates, we found that 326 women in a University cohort and 425 women in a health-maintenance organization cohort were at increased risks for UTI (2.57 [95% confidence interval (CI), 1.24-5.32] and 5.83 [95% CI, 3.17-10.70], respectively) if antimicrobials had been taken during the previous 15-28 days but not during the previous 3, 7, or 14 days. The increased risks were noted both for women whose antimicrobial use was for treatment of a previous UTI and for women who received antimicrobials for other illnesses. These results suggest that recent antimicrobial use increases a woman's risk of UTI, perhaps by altering the indigenous urogenital flora and predisposing to vaginal colonization with uropathogens.

Subhypnotic propofol does not treat postoperative vomiting in children after adenotonsillectomy.

PURPOSE: To investigate the efficacy of a subhypnotic dose of propofol to treat vomiting in children after adenotonsillectomy. METHODS: Two hundred and fifty-two children, aged 2-12 yr, underwent a standardized anaesthetic opioid administration, and postoperative care after adenotonsillectomy, adenoidectomy or tonsillectomy. A prospective, double-blinded, placebo-controlled study was performed in 70 of the patients who retched or vomited after surgery and who had intravenous access. Patients were assigned randomly to receive either 0.2 mg.kg-1) propofol (n = 35), or placebo (intralipid 10%, n = 35). RESULTS: The overall incidence of vomiting during the first 18-24 hr was 50%. Of those who had received propofol after the first episode of vomiting, 63% relapsed requiring a rescue antiemetic compared with 57% of those who had received intralipid (P = NS). Of the children who received propofol, 54% experienced pain on injection and 46% were mildly sedated compared with 3% and 11%, respectively, in the placebo group (P < 0.003). CONCLUSION: We conclude that an intravenous bolus of 0.2 mg.kg-1 propofolis not effective in the treatment of postoperative vomiting in children after adenotonsillectomy when a standardized anaesthetic with thiopentone, halothane, nitrous oxide, and 1.5 mg.kg-1 codeine phosphate is used, but it does cause sedation and pain on injection.

Partial enzymatic degradation of stroma allows enrichment and expansion of primary breast tumor cells.

The goal of this study was to isolate and expand tumor cells in culture that closely resemble invasive cells in primary breast carcinoma tissue. Based on the hypothesis that invasive tumor cells are released more readily upon digestion with connective tissue-degrading enzymes because they are not confined within a basement membrane, we have designed a novel procedure for their isolation. Using this method, we have successfully expanded in culture aneusomic tumor cells from several primary breast tumors. Twenty nine of 44 (66%) specimens processed yielded proliferative and passageable cultures of up to 2 x 10(7) cells. The original tumor tissue and cultures derived therefrom were compared for aneusomy and the abnormal expression of the erb-B2, p53, and bcl-2 gene products. Remarkable similarities were observed. However, some intratumor heterogeneity in chromosome content was found between touch preparations and cultured cells. Overexpression of erb-B2 was observed in the vast majority of cases (16 of 20), suggesting that this phenotype may be important for dysregulated proliferation in vitro. The simple and rapid method described in this report could enable routine expansion of primary breast tumors and provide adequate numbers of viable cells for studying and manipulating their functional characteristics.

Loss of heterozygosity in normal tissue adjacent to breast carcinomas.

Loss of heterozygosity (LOH) was detected in morphologically normal lobules adjacent to breast cancers. The most frequent aberration was at chromosome 3p22-25; of ten cases with this LOH in the carcinoma, six displayed the same LOH in adjacent normal lobules. This suggests that in a subset of sporadic breast cancers, a tumor suppresser gene at 3p22-25 may be important in initiation or early progression of tumorigenesis. Among sixteen breast cancers with LOH at 17p13.1 and five breast cancers with LOH at 11p15.5, one case each displayed the same LOH in adjacent normal lobules. Thus the molecular heterogeneity that characterizes invasive breast cancers may occur at the earliest detectable stages of progression.

An investigation of the current literature on the effectiveness of patient-controlled analgesia methods.

The purpose of this investigation was to determine, through current research in the literature, if a background basal infusion should routinely be used to improve the efficacy of traditional-demand patient-controlled analgesia (PCA) and would the safety of the PCA technique be maintained with the addition of a continuous infusion. Of the nine studies investigating PCA with and without continuous infusion, six found no improvement in pain control with the addition of a continuous infusion. The patients receiving continuous infusion did not make fewer demands than the control group, nor did they report lower pain scores. The addition of a continuous background infusion to PCA diminishes the inherent safety of the PCA modality of pain management. Many studies reported an increased incidence of side effects with the addition of a continuous infusion. This modality of PCA should be reserved for use in patients in whom traditional-demand PCA does not satisfy analgesic requirements.

Methylation of estrogen and progesterone receptor gene 5' CpG islands correlates with lack of estrogen and progesterone receptor gene expression in breast tumors.

Hormonal factors have a profound influence on the development, treatment, and outcome of breast cancer. The absence of steroid hormone receptors is highly correlated with resistance to antihormonal treatments. Work in cultured human breast cancer cell lines has shown that the absence of estrogen receptor (ER) gene expression in ER- cells is associated with extensive methylation of the ER gene 5' CpG island, and treatment with agents that demethylate the ER gene CpG island results in the production of functional ER protein. The current study shows that CpG islands in the 5' region of the ER and progesterone receptor (PR) genes are methylated in a significant fraction of primary human breast cancer tissues. The ER CpG island is methylated at the methylation-sensitive NotI restriction site in 9 of 39 (25%) of primary ER- breast cancers but remains unmethylated in 53 ER+ breast cancers and 9 normal breast specimens. Three methylation-sensitive restriction sites in the PR gene CpG island are not methylated in normal breast specimens and PR+ human breast cancers but are hypermethylated in 40% of PR- human breast tumors. These data demonstrate that methylation of the ER and PR gene CpG islands is associated with the lack of ER and PR gene expression in a significant fraction of human breast cancers.

Genetic analysis of breast cancer progression.

At the histological level, breast tumors display a variety of morphologic lesions which suggest the existence of an increasingly aberrant pathway of intermediate steps leading to the invasive primary tumor and its metastatic dissemination. In order to obtain direct evidence for this presumed progression, underlying genetic changes must be identified. Analyses of primary breast tumors have revealed a large number of dominant and recessive gene alterations encompassing several cellular attributes and activities. It is quite likely that some of these alterations are of a causal nature and thus enable the tumor to attain distinctive malignant phenotypes, such as, dysregulated proliferation, invasion, angiogenesis, and ability to metastasize. Considerable heterogeneity has been observed in the sequence of acquisition of these genetic changes, which is substantiated by recent comparative analyses between carefully microdissected preinvasive and invasive tumor. The data are evaluated here in the context of existing models of breast cancer progression. Implication and prospects for translational application to the clinic are also discussed.

Contiguous patches of normal human mammary epithelium derived from a single stem cell: implications for breast carcinogenesis.

Tissue clonality can be assessed in females by analyzing the methylation status of polymorphic DNA markers on X-linked genes because extensive de novo methylation of one allele at the preimplantation stage is associated with its permanent inactivation. We applied X chromosome inactivation toward understanding human breast morphogenesis by examining the nonmalignant breast epithelium from two reduction mammaplasties and a mastectomy. We found that entire lobules and large ducts of normal breast tissue have the same X chromosome inactivated, suggesting that they are derived from the same stem cell. The regions of inactivation of a particular X chromosome do not extend over an entire breast, so that ducts and lobules with opposite chromosomes inactivated are present within a single breast. Potential relevance of these observations for malignant transformation is discussed.