A genetic map of the mouse with 4,006 simple sequence length polymorphisms.

We have constructed a genetic map of the mouse genome containing 4,006 simple sequence length polymorphisms (SSLPs). The map provides an average spacing of 0.35 centiMorgans (cM) between markers, corresponding to about 750 kb. Approximately 90% of the genome lies within 1.1 cM of a marker and 99% lies within 2.2 cM. The markers have an average polymorphism rate of 50% in crosses between laboratory strains. The markers are distributed in a relatively uniform fashion across the genome, although some deviations from randomness can be detected. In particular, there is a significant underrepresentation of markers on the X chromosome. This map represents the two-thirds point toward our goal of developing a mouse genetic map containing 6,000 SSLPs.

Sphingosylphosphorylcholine is a ligand for ovarian cancer G-protein-coupled receptor 1.

Sphingosylphosphorylcholine (SPC) is a bioactive lipid that acts as an intracellular and extracellular signalling molecule in numerous biological processes. Many of the cellular actions of SPC are believed to be mediated by the activation of unidentified G-protein-coupled receptors. Here we show that SPC is a high-affinity ligand for an orphan receptor, ovarian cancer G-protein-coupled receptor 1 (OGR1). In OGR1-transfected cells, SPC binds to OGR1 with high affinity (Kd = 33.3 nM) and high specificity and transiently increases intracellular calcium. The specific binding of SPC to OGR1 also activates p42/44 mitogen-activated protein kinases (MAP kinases) and inhibits cell proliferation. In addition, SPC causes internalization of OGR1 in a structurally specific manner.

AKAP-mediated targeting of protein kinase a regulates contractility in cardiac myocytes.

Compartmentalization of cAMP-dependent protein kinase A (PKA) by A-kinase anchoring proteins (AKAPs) targets PKA to distinct subcellular locations in many cell types. However, the question of whether AKAP-mediated PKA anchoring in the heart regulates cardiac contractile function has not been addressed. We disrupted AKAP-mediated PKA anchoring in cardiac myocytes by introducing, via adenovirus-mediated gene transfer, Ht31, a peptide that binds the PKA regulatory subunit type II (RII) with high affinity. This peptide competes with endogenous AKAPs for RII binding. Ht31P (a proline-substituted derivative), which does not bind RII, was used as a negative control. We then investigated the effects of Ht31 expression on RII distribution, Ca(2+) cycling, cell shortening, and PKA-dependent substrate phosphorylation. By confocal microscopy, we showed redistribution of RII from the perinuclear region and from periodic transverse striations in Ht31P-expressing cells to a diffuse cytosolic localization in Ht31-expressing cells. In the presence of 10 nmol/L isoproterenol, Ht31-expressing myocytes displayed an increased rate and amplitude of cell shortening and relaxation compared with control cells (uninfected and Ht31P-expressing myocytes); with isoproterenol stimulation we observed decreased time to 90% decline in Ca(2+) but no significant difference between Ht31-expressing and control cells in the rate of Ca(2+) cycling or amplitude of the Ca(2+) transient. The increase in PKA-dependent phosphorylation of troponin I and myosin binding protein C on isoproterenol stimulation was significantly reduced in Ht31-expressing cells compared with controls. Our results demonstrate that, in response to beta-adrenergic stimulation, cardiomyocyte function and substrate phosphorylation by PKA is regulated by targeting of PKA by AKAPs.

Sixteen-week dose-intense chemotherapy in the adjuvant treatment of breast cancer.

Fifty-three women with breast cancer were treated with a new 16-week dose-intense, chemotherapy regimen. Patients with operable breast cancer with 10 or more histologically positive axillary nodes were treated with this five-drug regimen that incorporated the concepts of weekly chemotherapy, sequential administration of antimetabolites, and continuous infusion of fluorouracil (5-FU). The chemotherapy regimen consisted of eight cycles (each of 2 wk duration) of 100 mg of cyclophosphamide/m2 orally on days 1-7, 40 mg of doxorubicin/m2 intravenous (IV) on day 1, 100 mg of methotrexate/m2 IV on day 1 with 10 mg of leucovorin rescue/m2 every 6 hours for six oral doses on day 2, 1 mg of vincristine IV on day 1, and 600 mg of 5-FU/m2 IV at hour 20 over 2 hours. A continuous infusion of 300 mg of 5-FU/m2 per day was given IV on days 8-9 of each 2-week cycle. The doses and schedule of drug administration were designed to minimize dosage reduction and treatment delay. At a median follow-up of 17 months, there have been eight relapses in the 53 patients. The actuarial 3-year disease-free survival is 80% (95% confidence interval, 62% to 90%). The major side effects were attributable to myelosuppression. Absolute neutrophil counts less than 250/microL were noted in 12 (23%) patients; seven patients (13%) required hospitalization for management of neutropenic fever. No treatment-related deaths occurred. Ninety-four percent of the planned doses were administered, and only 5% of the courses were delayed because of toxic reactions. The encouraging therapeutic data, manageable side effects, and our ability to deliver over 90% of the planned doses provide the rationale for a phase III comparison of this new dose-intense regimen and standard chemotherapy in patients with operable disease and positive axillary nodes.

Effect of patients' expectations of benefit with standard breast cancer adjuvant chemotherapy on participation in a randomized clinical trial: a clinical vignette study.

Patients frequently overestimate the benefit of standard breast cancer adjuvant therapy. This is due in part to vague doctor-patient communication. To examine how the doctor's description and patient's expectations of the benefit of standard therapy affect clinical trial participation, we randomized 282 female cancer patients to one of two versions of a clinical vignette describing a choice between standard cyclophosphamide, methotrexate, and fluorouracil (5FU) (CMF) and a randomized trial comparing CMF with cyclophosphamide, doxorubicin, and 5FU (CAF). The vignettes differed only on whether results with CMF were described verbally or numerically in terms of disease-free survival (DFS). After selecting CMF or the trial, patients estimated their 10-year DFS with CMF. Patients were randomized 3:1 to the verbal vignette. The trial was selected by 110 of 210 (52.4%) verbal vignette patients versus 25 of 72 (34.7%) numeric vignette patients (P = .01). Estimates of 10-year DFS with CMF varied considerably; many were inaccurate. When patients in the verbal vignette group were divided into thirds according to DFS estimate, 22 of 64 (34.4%) in the top third selected the trial versus 38 of 64 (59.4%) and 38 of 65 (58.5%) in the middle and bottom third, respectively (P = .005). Younger age, college education, and previous participation in a trial also predicted trial selection. Multivariate logistic regression suggested that the benefit expected from CMF was more important than how benefit was described in treatment selection. Assuring realistic patient expectations of standard adjuvant therapy benefit is likely to be important during discussion of clinical trials.

A new biomarker in monitoring breast cancer: CA 549.

Serum biomarkers are not very reliable in assessing outcome or predicting recurrence of breast cancer. Clinically, carcinoembryonic antigen (CEA) is widely used and is elevated in a majority of patients with metastatic breast cancer. However, it is falsely elevated in a wide range of nonmalignant conditions and correlates poorly with disease progression. We evaluated a newly described monoclonal antibody, CA 549, in an immunoradiometric assay which uses two monoclonal antibodies directed against tumor and milk fat globule membranes. CA 549 and CEA were studied in 682 patients, 331 of whom had breast diseases and 99 of whom were followed with multiple serum samples. Of 69 patients with benign breast diseases, 1.5% had elevated CA 549, 0% of 30 pregnant women had elevated CA 549, and 26% of patients with nonmalignant liver disease had CA 549 elevation. In metastatic cancer of prostate, ovary, endometrium, colon, and lung CA 549 was elevated in 12% to 50% of cases with levels less than 120 U/mL. In breast cancer, CA 549 was elevated in 11% of 88 patients who received adjuvant chemotherapy and had no evidence of metastasis; in 23% of 16 patients in complete remission after chemotherapy; in 63% of 52 patients in partial remission after therapy; and in 83% of 106 patients with progression of breast cancer compared with 63% with elevated CEA (P = .001). In diseases of the breast, CA 549 has a sensitivity In diseases of the breast, CA 549 has a sensitivity and specificity of 77% and 92% v 61% and 92% for CEA. Of 99 patients serially monitored with clinically documented breast cancer progression, regression, or stability of disease, CA 549 was statistically significantly superior to CEA in monitoring a greater than 25% change in those patients with metastatic progression (P = .03). CA 549 is a new serum marker that should be control tested in prospective clinical trials alone or in conjunction with other markers.

Once-daily irrigation of long-term urethral catheters with normal saline. Lack of benefit.

Urinary incontinence is often managed with a long-term urethral catheter after other methods have failed. Such urethral catheterization is associated with polymicrobial bacteriuria, catheter obstruction, fever, bacteremia, urinary tract stones, and death. Periodic catheter irrigation is a common but untested management procedure intended to prevent catheter obstruction, fevers, and/or bacteremia. To evaluate this technique, we performed a randomized crossover trial comparing ten weeks of once-daily normal saline irrigation with ten weeks of no irrigation in 32 long-term catheterized women. The incidence of catheter obstructions and febrile episodes and the prevalence and species of bacteriuria were similar whether examined as completed crossover patients (N = 23) or as partially completed trials (N = 9). Once-daily irrigation with normal saline of long-term urethral catheters is a time-consuming and costly procedure that is unlikely to have an impact on the morbidity associated with such catheters.

Commonly used methods of estimating creatinine clearance are inadequate for elderly debilitated nursing home patients.

We evaluated the Jelliffe and the Cockcroft and Gault methods of estimating creatinine clearance in elderly nursing home patients (65 years) with chronic indwelling urethral catheters. Although these relationships have been prospectively validated in hospitalized and ambulatory populations previously, we found that they produced poor estimates of creatinine clearance in this patient group. For the Jelliffe method, 11 of 19 estimates were 20% or greater from the measured value; 10 of 11 poor estimates were high. The Cockcroft-Gault method had 10 of 19 estimates 20% or greater from the measured value with 8 of 10 of the poor estimates being high. An altered relationship between body weight, muscle mass, and daily creatinine production is the most likely explanation for the bias in these creatinine-clearance estimations. New estimates of the relationships between age, weight, serum creatinine, and creatinine clearance need to be developed for this population.

Changes in umbilical arterial and venous blood flow velocity waveforms during late decelerations of the fetal heart rate.

OBJECTIVE: To compare umbilical arterial and venous Doppler velocity waveforms during labor in fetuses with normal heart rate tracings and fetuses with late decelerations. METHODS: During labor, umbilical arterial and venous Doppler flow velocity waveforms were obtained between and during contractions in 20 fetuses (ten with normal heart rate tracings and ten with late decelerations). The umbilical arterial systolic-diastolic (S/D) velocity ratios were compared, and umbilical venous velocities were examined for the presence or absence of venous pulsations. RESULTS: Fetuses with late decelerations had a significant increase in the umbilical arterial S/D ratio between contractions compared to normal fetuses (2.43 +/- 0.74 versus 1.56 +/- 0.27, P = .003). There was no significant difference in S/D ratios obtained between contractions compared with those obtained during contractions in either the normal or late deceleration group. Umbilical venous pulsations were significantly more common (90 versus 0%, P < .00001) during contractions in fetuses with late decelerations; umbilical venous pulsations were not seen in these fetuses between contractions and were not present at any time in fetuses with normal heart rate tracings. CONCLUSIONS: Increased umbilical arterial velocity ratios and the presence of umbilical venous pulsations during contractions in fetuses with late decelerations suggest that the cardiovascular pathophysiology of late decelerations involves changes in placental resistance as well as cardiac performance. The presence of umbilical venous pulsations during late decelerations is consistent with the hypoxic fetal cardiovascular changes (hypertension and myocardial depression) previously reported from animal studies.

Effects of calcium antagonists on the evoked release of dynorphin A(1-8) and availability of intraterminal calcium in rat hippocampal mossy fiber synaptosomes.

The pharmacological properties of presynaptic calcium (Ca) channels on rat hippocampal mossy fiber synaptosomes were characterized by determining the inhibitory potencies for various classes of Ca antagonists on depolarization-induced Ca mobilization and the release of dynorphin A(1-8)-like immunoreactivity (Dyn-LI). Flunarizine and cinnarizine were the most potent inhibitors of both parameters (IC50 values less than 10(-5) M). Gadolinium and omega-conotoxin (omega-CgTx) were also effective inhibitors of Dyn-LI release (IC50 values less than 3 x 10(-5) M), but omega-CgTx only partially reduced the level of cytosolic free Ca. The release of Dyn-LI was relatively insensitive to both the L-type (dihydropyridines, verapamil and diltiazem) and T-type (amiloride and phenytoin) channel blockers. It appears that presynaptic N-type Ca channels make the most substantial contribution to the Ca influx required for the exocytosis of Dyn-LI from hippocampal mossy fiber nerve endings.

Factors associated with fruit and vegetable consumption among women participating in WIC.

OBJECTIVE: The purpose of this analysis was to assess to what extent sociodemographic and psychosocial characteristics predict consumption of fruits and vegetables in women served by the Special Supplemental Nutrition Program for Women, Infants, and Children (WIC). DESIGN: Baseline survey data were analyzed for women enrolled in the Maryland WIC 5 A Day Promotion Program, a study sponsored by the National Cancer Institute. Sociodemographic variables included age, race, education, marital status, working status, pregnancy status, and smoking status. Psychosocial variables included self-efficacy, knowledge, attitudes, social support, and perceived barriers to consuming 5 or more servings daily of fruits and vegetables. SUBJECTS/SETTING: Analyses are based on 3,122 women enrolled at 15 WIC sites in Baltimore and 6 counties in Maryland. STATISTICAL ANALYSES PERFORMED: Because psychosocial characteristics were measured on different scales and varied in dispersion, we standardized scores for these variables. Multiple regression analyses were then performed to assess contributions of the sociodemographic variables and the standardized psychosocial variable scores to the variance in consumption of fruits and vegetables. RESULTS: Sociodemographic variables were not powerful predictors of fruit and vegetable consumption. In contrast, an increase of 1 standard deviation in self-efficacy resulted in a mean increase of 0.76 servings, and an increase of 1 standard deviation in perceived barriers resulted in a decrease of 0.50 servings. About 21% of the variance in consumption was explained by all of the variables examined. APPLICATIONS: Dietitians and intervention programs should focus on increasing clients' self-efficacy, positive attitudes, and knowledge relating to fruits and vegetables while trying to reduce perceived barriers to consumption of fruits and vegetables.

Calcium mobilization in ovarian cancer cells in response to lysophospholipids.

Intracellular free Ca(2+)concentration ([Ca(2+)](i)) plays a critical role in regulating many diverse cellular functions including cell proliferation and programmed cell death (apoptosis) (1). An elevation in [Ca(2+)](i) activates enzymes (phospholipase A(2), phospho- lipase D and some isoforms of protein kinase C) associated with the liberation of bioactive lipids such as arachidonic acid (AA) and lysophosphatidic acid (LPA) (1,2). AA and its metabolites have been implicated in multiple steps of carcinogenesis (3,4). LPA as well as several other bioactive lipids stimulate release of Ca(2+)from intracellular stores and regulate proliferation of ovarian cancer cells I5-7).

Factors associated with attendance in a voluntary nutrition education program.

PURPOSE: This paper examines factors associated with attendance in a National Cancer Institute-funded randomized trial of nutrition education to increase fruit and vegetable consumption among women served by the Special Supplemental Nutrition Program for Women, Infants, and Children (WIC). SETTING: The study took place at 16 WIC sites in Maryland. SUBJECTS: The participants were 1528 women who were enrolled in WIC or who had children enrolled in WIC, were > or = 18 years of age, and planned to continue enrollment at that WIC site for at least 6 months (68% of eligible women approached agreed to participate). INTERVENTION: Women received personal invitations, letters, and telephone reminders from peer educators encouraging their attendance at three bimonthly nutrition sessions. MEASURES: Demographic data were collected in a baseline survey. Attendance data and telephone and address changes were also collected. The postintervention survey included a question regarding reasons for nonattendance. Focus groups were also held to ascertain reasons for attendance or nonattendance. Chi-square tests of trend and multiple logistic regression, adjusted for within-site correlation, were used in statistical analyses. RESULTS: Fifty-four percent of enrollees attended at least one session. Multiple logistic regression analysis showed increased odds of attending with higher age, breast-feeding, and/or knowledge of the recommendation to eat five or more servings of fruits and vegetables daily. There were decreased odds of attending for pregnant women who already had children. There were nonsignificant trends toward decreased attendance among unmarried women compared with married women and among blacks compared with nonblacks. Reasons given for nonattendance included withdrawal from WIC, moving, conflicting activities, negative feelings about nutrition education, and lack of transportation or child care. CONCLUSIONS: The results suggest that numerous barriers hinder participation in nutrition programs aimed at low-income women. These barriers should be considered by health care professionals when planning intervention programs. Overcoming these barriers presents a major challenge.

5 A Day for better health--nine community research projects to increase fruit and vegetable consumption.

One of the national objectives in "Healthy People 2000" is for members of the public to increase their consumption of fruits and vegetables to five or more servings daily. The National Cancer Institute (NCI) began a nationwide campaign to achieve this objective in 1991. As part of this campaign, the NCI funded nine research studies in 1993. These projects are implementing and evaluating community-based programs designed to increase fruit and vegetable consumption among different segments of the population in Alabama, Arizona, Georgia, Louisiana, Maryland, Massachusetts, Minnesota, North Carolina, and Washington. The settings for these projects include the Special Supplement Food Program for Women, Infants, and Children (WIC Programs), churches, worksites, and schools. The projects are led by multidisciplinary teams and entail extensive collaboration among academic, governmental, private sector, and voluntary agencies within each State. The projects represent a model public health paradigm for conducting this type of research.

Uses of process evaluation in the Maryland WIC 5-a-day promotion program.

Using a crossover design, the authors conducted a 6-month intervention program aimed at increasing fruit and vegetable consumption among women served by the Women, Infants, and Children program in Baltimore City and six Maryland counties. The theoretical framework for the interventions was the Transtheoretical Model of Change. At 2 months postintervention, mean daily consumption had increased significantly more in intervention participants than in control participants. Extensive process evaluation data were collected to assess the quantity and quality of program services delivered. These included participant nutrition session evaluation forms and attendance logs, focus groups of attenders and nonattenders of sessions, information about peer educators, and postintervention surveys. Many lessons were learned about program delivery, factors affecting attendance, and the obstacles to dietary change. Strategies to increase participants' consumption of fruits and vegetables were modified based on these lessons and the process evaluations.

The effect of the Maryland WIC 5-A-Day promotion program on participants' stages of change for fruit and vegetable consumption.

The Maryland Women, Infants and Children (WIC) 5-A-Day Promotion Program examined the effect of a multifaceted nutrition intervention on changing the fruit and vegetable consumption of low-income women in the WIC program in Maryland. The sample consisted of 3,122 participants (1,443 intervention and 1,679 control) with a mean age of 27.2. Fifty-six percent were Black/African American. This article focuses on the effect of the intervention on the stages of change of the participants. Intervention participants showed significantly greater positive movement through the stages than control participants. Stages of change were measured for two specific target behaviors: eating five servings of fruits and vegetables a day and eating more servings of fruit and vegetables a day. Results demonstrated significant differences in the stage status of intervention and control women and in movement through the stages. The effectiveness of the intervention across groups depended on which staging measure was used.

Modulation of glutamate release from hippocampal mossy fiber nerve endings by arachidonic acid and eicosanoids.

Arachidonic acid has been implicated in normal synaptic transmission processes, including those related to the development of hippocampal long-term synaptic potentiation. Hippocampal mossy fiber (MF) synaptosomes were used to investigate the role of arachidonate in the evoked accumulation of presynaptic Ca2+ and the release of endogenous glutamate, since these nerve terminals express long-term potentiation and selectively release glutamate as the excitatory transmitter. It was demonstrated that membrane depolarization evoked the accumulation of Ca2+, the release of glutamate, and the production of unesterified arachidonic acid. These events may be functionally related, since exogenous arachidonate and phospholipase A2 activation mimicked the effects of depolarization on Ca2+ availability and glutamate release, while secretion processes were attenuated in the presence of phospholipase A2 inhibitors. In addition, pretreatment of the nerve terminals with arachidonate or melittin allowed for the facilitated release of glutamate in response to a subsequent depolarizing stimulus. Inhibition of cyclooxygenase or lipoxygenase activities also potentiated presynaptic responses to membrane depolarization. In contrast, 12-lipoxygenase products attenuated the depolarization-evoked accumulation of intraterminal free Ca2+ and glutamate release. It is suggested that arachidonic acid acts as a positive modulator of mossy fiber secretion processes, including those involved in the increased glutamate release required for the induction of long-term potentiation, while 12-lipoxygenase metabolites provide negative feedback signals designed to limit neurotransmitter secretion.

[3H]arachidonic acid metabolism in rat brain minces: effects of nucleotide triphosphates, CDPcholine and CMP.

Rat brain minces were used to investigate the effects of nucleotides on the metabolism of arachidonic acid in nerve tissue. Brain free fatty acids, neutral lipids and phospholipids, were radiolabeled in vivo following intracerebral injection of [3H]arachidonic acid. Minces were prepared from the radiolabeled cerebra and were incubated in a modified Krebs-Ringer buffer with and without various nucleotides. The incubation-induced accumulation of unesterified [3H]arachidonate was reduced in the presence of CDPcholine, ATP, CTP, GTP, and UTP. These nucleotides inhibited choline and inositol glycerophospholipid hydrolysis. They also reduced the amount of labeled diglycerides. However, CDPethanolamine had no effect on arachidonic acid metabolism in the mince preparation and CMP appeared to stimulate further hydrolysis of choline glycerophopholipids, resulting in increased accumulation of [3H]arachidonic acid and labeled diglycerides. We suggest that the production of unesterified [3H]arachidonate and labeled diglycerides is due to the involvement of more than one catabolic reaction, since the high energy nucleotides had similar effects on fatty acid accumulation, but different effects on phospholipid labeling.

Arachidonic acid enhances contraction and intracellular Ca2+ transients in individual rat ventricular myocytes.

Modulation of intracellular free Ca2+ concentration ([Ca2+]i) by inotropic stimuli alters contractility in cardiac muscle. Arachidonic acid (AA), a precursor for eicosanoid formation, is released in response to receptor activation and myocardial ischemia and has been demonstrated to alter K+ and Ca2+ channel activity. We investigated the effects of AA on contractility by simultaneously measuring [Ca2+]i and shortening in single field-stimulated rat ventricular myocytes. [Ca2+]i transients were measured using fura 2, and myocyte shortening was assessed using video edge detection. AA stimulated a doubling in the amplitude of the [Ca2+]i transient and a twofold increase in myocyte shortening. In addition, AA stimulated a 30% increase in the time to 50% diastolic [Ca2+]i and a 35% increase in the time to 50% relengthening. These effects of AA were mediated by AA itself (56 +/- 5%) and by cyclooxygenase metabolites. Pretreatment with the protein kinase C inhibitors staurosporine and chelerythrine nearly abolished (> 90% inhibition) these AA-induced effects. Inhibition of voltagegated K+ channels with 4-aminopyridine mimicked the effects of AA. Addition of AA to the 4-aminopyridine-treated myocyte had no additional effect on parameters of contractile function. These data indicate that AA alters the amplitude and duration of Ca2- transients and myocyte shortening via protein kinase C-dependent inhibition of voltage-gated K+ channels. Release of AA by phospholipases in response to receptor activation by endogenous mediators or pathological stimuli may be involved in mediating inotropic responses in cardiac muscle.