Factors affecting Agrobacterium tumefaciens-mediated transformation of peppermint.

Substantial improvement in peppermint (Mentha x piperita L. var. Black Mitcham) genetic transformation has been achieved so that the frequency of transgenic plants regenerated (percent of leaf explants that produced transformed plants) was 20-fold greater than with the original protocol. Essential modifications were made to conditions for Agrobacterium tumefaciens co-cultivation that enhanced infection, and for selection of transformed cells and propagules during regeneration. A systematic evaluation of co-cultivation parameters established that deletion of coconut water from the co-cultivation medium resulted in substantially increased transient ß-Glucuronidase (GUS) activity, in both the frequency of explants expressing gusA and the number of GUS foci per explant (>700 explants). Co-cultivation on a tobacco cell feeder layer also enhanced A. tumefaciens infection. Enhanced transformation efficiencies were further facilitated by increased selection pressure mediated by higher concentrations of kanamycin in the medium during shoot induction, regeneration, and rooting: from 20 to 50 mg/l in shoot induction/regeneration medium and from 15 to 30 mg/l in rooting medium. Raising the concentration of kanamycin in media substantially lowered the number of "escapes" without significant reduction in plant regeneration. These modifications to the protocol yielded an average transformation frequency of about 20% (>2000 explants) based on expression of GUS activity or the tobacco antifungal protein, osmotin, in transgenic plants. Genetic transformation of peppermint has been enhanced to the extent that biotechnology is a viable alternative to plant breeding and clonal selection for improvement of this crop.

The effectiveness of condoms in reducing heterosexual transmission of HIV.

CONTEXT: It is not established whether the condom is as effective at preventing heterosexual transmission of HIV as it is for preventing conception. An overall estimate of condom effectiveness for HIV prevention is needed. METHODS: Information on condom usage and HIV serology was obtained from 25 published studies of serodiscordant heterosexual couples. Condom usage was classified as always (in 100% of acts of intercourse), sometimes (1-99%, 0-99% or 1-100%) or never (0%). Studies were stratified by design, direction of transmission and condom usage group. Condom efficacy was calculated from the HIV transmission rates for always-users and never-users. RESULTS: For always-users, 12 cohort samples yielded a consistent HIV incidence of 0.9 per 100 person-years (95% confidence interval, 0.4-1.8). For 11 cohort samples of never-users, incidence was estimated at 6.8 per 100 person-years (95% confidence interval, 4.4-10.1) for male-to-female transmission, 5.9 per 100 (95% confidence interval, 1.5-15.1) for female-to-male transmission and 6.7 per 100 (95% confidence interval, 4.5-9.6) in samples that specified the direction of transmission. Generally, the condom's effectiveness at preventing HIV transmission is estimated to be 87%, but it may be as low as 60% or as high as 96%. CONCLUSIONS: Consistent use of condoms provides protection from HIV. The level of protection approximates 87%, with a range depending upon the incidence among condom nonusers. Thus, the condom's efficacy at reducing heterosexual transmission may be comparable to or slightly lower than its effectiveness at preventing pregnancy

PIP: This cross-sectional and longitudinal study examines the overall effectiveness of condoms in reducing heterosexual transmission of HIV. Data on condom usage and HIV serology was obtained from 25 published studies of serodiscordant heterosexual couples. Condom usage was rated as ¿always¿ (in 100% of acts of intercourse), ¿sometimes¿ (1-99%, 0-99%, or 1-100%), or ¿never¿ (0%). Studies were stratified by design, direction of transmission and condom usage group. Condom efficacy was calculated from the HIV transmission rates for always-users and never-users. Results showed that 12 cohort samples gave a consistent HIV incidence of 0.9 per 100 person-years. For 11 cohort samples of never-users, incidence was estimated at 6.8 per 100 person-years for male-to-female transmission and 5.9 per 100 for female-to-male transmission. Overall, the condom's effectiveness at preventing HIV transmission is estimated to be 87%, but it may vary between 60% and 96%.

Consensus theory model of AIDS/SIDA beliefs in four Latino populations.

To describe Latino beliefs about AIDS (SIDA), Latino adults were sampled at two U.S. sites (Connecticut and Texas) and two international sites (Mexico and Guatemala). A 125-item questionnaire covered risk factors, symptoms, treatments, and sequellae of AIDS. The cultural consensus model was used to determine the cultural beliefs for each sample. Responses from 161 people indicated that a single set of beliefs was present at each site and that beliefs were shared across sites. Comparison of answers between samples indicated high agreement (p < .0007). The proportion of shared beliefs, however, decreased significantly between samples: .68 in Connecticut, .60 in Texas, .51 in Mexico, and .41 in Guatemala (p < .05). The proportion of positive answers similarly decreased from Connecticut to Guatemala (p < .001). Beliefs were stronger and more detailed in the higher prevalence areas. Furthermore, Latino beliefs tended to converge on biomedical beliefs about the disease.

Rapid ethnographic assessment of breastfeeding practices in periurban Mexico City.

Before carrying out a breastfeeding promotion programme in a periurban area of Mexico City, we conducted a rapid ethnographic study to determine the factors associated with absence of exclusive breastfeeding. The responses to pilot interviews were used to develop a standardized questionnaire regarding reasons for infant feeding choice, sources of advice, and barriers to breastfeeding. We interviewed a random sample of 150 mothers with a child < 5 years of age; 136 (91%) of them had initiated breastfeeding; but only 2% exclusively breastfed up to 4 months. The mothers consistently stated that the child's nutrition, health, growth, and hygiene were the main reasons for the type of feeding selected; cost, comfort, and the husband's opinion were less important. Physicians were ranked as the most important source of advice. Reduction or cessation of breastfeeding occurred on the doctor's advice (68%); or when the mothers encountered local folk illnesses such as "coraje" (52%) or "susto" (54%), which are associated with anger or fright; or had "not enough milk" (62%) or "bad milk" (56%); or because of illness of the mother (56%) or child (43%). During childhood illnesses and conditions, breastfeeding was reduced and the use of supplementary foods was increased. This study emphasizes the importance of cultural values in infant feeding choices, defines specific barriers to breastfeeding, and provides a basis for interventions to promote exclusive breastfeeding in the study population.

PIP: Prior to initiating a community-based intervention program to promote exclusive breast feeding in San Pedro Martir, Mexico, a 2-month (1994) rapid ethnographic assessment was conducted. 150 mothers whose youngest child was under 5 years of age were interviewed. 136 mothers (91%) had breast-fed their infant, for a median duration of 6 months, but only 2% exclusively breast-fed for up to 4 months. Mothers consistently described breast feeding as the best nutrition for their infant. However, the dominant feeding pattern was mixed breast and bottle-feeding. Formula, tea, and water were introduced during the first postpartum day. By the end of the third month, 63% of mothers had introduced solid food to promote growth. It was common practice to reduce breast feeding and increase feeding of supplementary foods when a child was ill. Physicians were the most respected source of knowledge on breast feeding. 42% of mothers reported that, at some point when they were breast feeding, a doctor had advised them to stop and half these mothers complied. The data collected in this rapid survey were used to guide a peer counseling program to promote exclusive breast feeding in the community.

Latino beliefs about diabetes.

OBJECTIVE: To describe Latino beliefs about diabetes and assess heterogeneity in beliefs across different groups. RESEARCH DESIGN AND METHODS: This study comprised a survey of 161 representative Latino adults from four diverse communities: Hartford, Connecticut; Edinburg, Texas; Guadalajara, Mexico; and rural Guatemala. A 130-item questionnaire covered causes symptoms, and treatments for diabetes. Information on demographics and acquaintanceship with someone with diabetes was also collected. The cultural consensus model was used to analyze the variation in responses to determine whether the degree of consistency within and between samples was sufficient to warrant aggregation and description as a single set of beliefs. RESULTS: Homogeneous beliefs were present within each of the four samples. Although variability in responses increased significantly from Connecticut to Guatemala (P < 0.00005), there was significant agreement between samples on the answers (P < 0.0005). Answers tended to be concordant with the biomedical description of diabetes. Greater acculturation, higher educational attainment, and higher diabetes prevalence were associated with greater cultural knowledge about diabetes. In Connecticut, greater knowledge correlated with longer mainland U.S. residency (P < 0.05). In Mexico, those with average educational attainment knew more (P < 0.05). Finally, average knowledge levels were higher in communities with greater diabetes prevalence. CONCLUSIONS: The cultural consensus model facilitated assessment of cultural beliefs regarding diabetes and diabetes management. Overall, Latino cultural beliefs about diabetes were concordant with the biomedical model. Variation in responses tended to characterize less knowledge or experience with diabetes and not different beliefs.

Transgenic peppermint (Mentha×piperita L.) plants obtained by cocultivation with Agrobacterium tumefaciens.

The first transgenic peppermint (Mentha×piperita L. cultivar Black Mitcham) plants have been obtained by Agrobacterium-mediated transformation by cocultivation with morphogenically responsive leaf explants. Basal leaf explants with petioles, from leaves closest to the apex of in-vitro-culture-maintained shoots (5 cm), exhibited optimal shoot organogenetic responsiveness on medium supplemented with thidiazuron (8.4 µM). Shoot formation occurred at sites of excision on the leaf blade and petiole either directly from cells of the explant or via a primary callus. Analyses of transient GUS activity data indicated that DNA delivery by microprojectile bombardment was more effective than Agrobacterium infection. However, no transgenic plants were obtained from over 22,000 leaf explants after particle bombardment. Cocultivation of leaf explants with Agrobacterium strain EHA 105 and kanamycin selection produced transgenic plants. Greater transient and stable -glucuronidase (GUS) activities were detected in explants or propagules transformed with the construct where gusA was driven by the pBISN1 promoter rather than a CaMV 35S promoter. Eight plants were subsequently regenerated and verified as transgenic based on detection of the nptII transgene by PCR and Southern blot analyses. The Southern analyses indicated that the plants were derived from eight unique transformation events. All transgenic plants appeared morphologically normal. Analyses of GUS activities in leaves sampled from different portions of these transgenic plants, 10 months after transfer to the greenhouse, indicated that six out of the eight original regenerants were uniformly transformed, i.e., did not exhibit chimeric sectors.

Predicting treatment-seeking behavior in Guatemala: a comparison of the health services research and decision-theoretic approaches.

This study attempts to identify and describe factors associated with the choice of a health care source in rural Guatemala. Because of limited choice options, rural Guatemala makes an excellent location for studying the factors that affect utilization patterns. Illness case histories were collected from a random sample of 270 households in six villages. Then, two different methodological approaches were used to predict treatment actions. First, a sociobehavioral model, which encompasses enabling, predisposing, and need factors, was used to predict treatment choices. Using discriminant analysis we identified factors associated with the use of home remedies, a pharmacy, the health post, a physician, or folk healer. In a second, parallel study, descriptive interviews were used to identify important factors in choosing a treatment strategy. From these interviews, and from responses to hypothetical illness cases, we developed a decision model of treatment actions. Both models were tested against the set of illness cases. Results indicate that both approaches identify similar variables (especially, severity), although selection of variables through the multivariate analysis was much more successful in predicting treatment actions.

Assessing rater performance without a "gold standard" using consensus theory.

This study illustrates the use of consensus theory to assess the diagnostic performances of raters and to estimate case diagnoses in the absence of a criterion or "gold" standard. A description is provided of how consensus theory "pools" information provided by raters, estimating rater competencies and differentially weighting their responses. Although the model assumes that raters respond without bias (i.e., sensitivity = specificity), a Monte Carlo simulation with 1,200 data sets shows that model estimates appear to be robust even with bias. The model is illustrated on a set of elbow radiographs, and consensus-model estimates are compared with those obtained from follow-up data. Results indicate that with high rater competencies, the model retrieves accurate estimates of competency and case diagnoses even when raters' responses are biased.

An asthma self-management program for children, including instruction in peak flow monitoring by school nurses.

A randomized trial of an instructional method was conducted in which school nurses taught children asthma self-management principles and skills, including peak flow monitoring, in 20-min, individual sessions over an 8-week period. Thirty-six children participated. An intervention group of 18 children received the teaching sessions. A control group of 18 children received regular care by the nurses, but no teaching sessions. The sample included 64% boys, 69% African-Americans, and 69% Medicaid recipients. The average age of subjects was 10.2 years. The two groups were demographically similar, but despite random assignment, the control group had a significantly earlier age of onset of asthma and tended to have had more asthma attacks in the preceding year. These factors were statistically controlled in outcome analyses. Results of group comparisons showed no significant differences in the number of postintervention emergency room visits and days absent from school. However, nurses reported that children who practiced breathing exercises had less anxiety during exacerbations, and the nurses' knowledge of the children's baseline peak expiratory flow rates facilitated care of the children. Nurses expressed the opinion that the individual sessions with students might be useful in motivating them to participate effectively in later group sessions. The intervention was well accepted by students, parents, and nurses. We believe that this intervention is promising as a practical, low-cost approach to enhancing children's asthma self-management skills and warrants further testing in a larger sample, with the intervention conducted over a longer period.

Stability and expression of amplified EPSPS genes in glyphosate resistant tobacco cells and plantlets.

The stability and expression of amplified 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) genes was examined in glyphosate resistant tobacco cells grown in glyphosate-free medium, and in plantlets regenerated from resistant cells. Amplified DNA was maintained in resistant cells grown in the absence of glyphosate for three years. Amplified EPSPS genes were retained in regenerated plantlets at levels comparable to those observed in the resistant cells, and EPSPS mRNA was overexpressed (compared to unselected plantlets). However, glyphosate resistance in cell lines grown in glyphosate-free medium declined 7-fold, and in regenerated plantlets approximately 20-fold, compared to resistant cells maintained under glyphosate selection. In plantlets, reduced resistance correlated with lower levels of EPSPS mRNA. Plantlets regenerated from resistant cells exhibited morphological variation, and had an approximate doubling of their nuclear genome size.

Impaired Wound Induction of 3-Deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) Synthase and Altered Stem Development in Transgenic Potato Plants Expressing a DAHP Synthase Antisense Construct.

Potato (Solanum tuberosum L.) cells were transformed with an antisense DNA construct encoding part of 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase (EC 4.1.2.15), the first enzyme of the shikimate pathway, to examine the role(s) of this protein in plant growth and development. Chimeric DNA constructs contained the transcript start site, the first exon, and part of the first intron of the shkA gene in antisense or sense orientations under the control of the cauliflower mosaic virus 35S promoter. Some, but not all, of the transgenic plants expressing antisense DAHP synthase RNA showed reduced levels of wound-induced DAHP synthase enzyme activity, polypeptide, and mRNA 12 and 24 h after wounding. No alteration in the wound induction of DAHP synthase gene expression was observed in transgenic potato tubers containing the chimeric sense construct. Reduced steady-state levels of DAHP synthase mRNA were observed in stem and shoot tip tissue. Some plants with the chimeric antisense construct had reduced stem length, stem diameter, and reduced stem lignification.

Knowledge, beliefs, and practices in relation to malaria transmission and vector control in Guatemala.

As part of an effort to involve community members in malaria control activities, we studied knowledge, beliefs, and practices of residents of both the Pacific coastal plain and northeastern Guatemala related to malaria transmission and Anopheles albimanus control. Most residents recognized the role of mosquitoes in malaria transmission, but few knew how mosquitoes acquired their infections or understood the risk of having an untreated person in their midst. If this were more widely known, residents might put greater pressure on infected patients to seek timely and appropriate antimalarial treatment. Seventy-three percent of families owned one or more bed nets; however, even though most informants believed that bed nets help protect against malaria, the major reason for using them was to prevent nuisance mosquito bites. It is concluded that efforts should be made to promote bed net use by seeking ways to make them more affordable and by emphasizing their effectiveness as a barrier to nuisance mosquitoes. Although residents have a very positive opinion of the National Malaria Service spray teams, it is proposed that cooperation might be improved if malaria workers would emphasize the fact that house spraying reduces the numbers of nuisance mosquitoes and other pest insects, rather than focusing solely on malaria prevention, which most informants believed was less important. This study emphasizes the importance of understanding community beliefs and practices when planning or evaluating vector control activities.

Predictors of lead stores in male veterans.

Thirty-two male veterans participated in a study to determine cumulative lead exposure in an urban population. Subjects were chosen on the basis of blood pressure status in order to attempt to compare lead exposure between patients with and without hypertension. Patients currently enrolled in hypertension clinic and on treatment were recruited and matched with controls for age, race, and socioeconomic status. Each subject underwent provocative chelation via slow intravenous infusion of CaNa2 EDTA and 6-h urinary lead measurement and completed an interviewer-administered questionnaire. Twenty blacks and 12 whites participated, with a median age of 52 years (range: 27 to 72). Urinary lead excretion ranged from below detection limits to frankly toxic levels in an individual with heavy moonshine ingestion. Lead levels were higher than reported in other non-workplace populations. The distribution of lead values was skewed, as expected, with a median excretion of 75 mcg lead/6 h (corresponding to a median 24 degrees post-chelation urinary lead excretion of 286 mcg) and modal values between 50 and 75 micrograms lead. Levels of 95 mcg lead/6 h (corresponding to 24 degrees levels of 333 mcg lead) and above were considered "high" (N = 11) and the remainder were "low" (N = 21). Among those able to recall various characteristics of their first childhood dwellings, the presence of flaking paint in a multiple family dwelling was strongly associated with "high" lead excretion (X2 = 9.32, p = 0.009). Hypertensives excreted slightly more lead than nonhypertensives, although the difference was not statistically significant in this small sample. Lead excretion was not associated with current (treated) blood pressure determinations among hypertensives. However, lead excretion was associated with systolic pressure as recorded on entry to the hypertension clinic (N = 21, R2 = 0.24, p = 0.03).

Qualities of an ideal volunteer community malaria worker: a comparison of the opinions of community residents and national malaria service staff.

Since the late 1950s, most malaria surveillance and treatment in rural areas of Latin America has been carried out by networks of unpaid community malaria workers, known as Volunteer Collaborators, who are selected and supervised by staff of the national malaria services (NMSs) in each country. In spite of the free and readily accessible antimalarial treatment available at these Volunteer Collaborator posts, many residents continue to seek treatment elsewhere and in most cases take doses of antimalarials that are insufficient to cure their infections. To identify ways in which the Volunteer Collaborator Network could be made more attractive to residents and to improve the process of selection of new workers, we asked community residents and Guatemalan NMS workers to rank order, according to their importance, 11 qualities or characteristics of an 'ideal' volunteer malaria worker. Community residents preferred someone who is available to take care of patients at all times of the day, is a responsible person, and has a general knowledge of medicine. No significant differences were noted in the rank orders of male and female residents or literate and illiterate residents. National Malaria Service workers also preferred someone who takes care of patients at all times of the day, even when busy. In addition, they wanted individuals who recognize the importance of their work as a Volunteer Collaborator, but choosing volunteers who had a general knowledge of medicine was not important. By modifying the procedures used to select Volunteer Collaborators so as to identify candidates with the qualities preferred by residents, it should be possible to increase acceptance and improve the performance of these volunteer workers.

Obesity, school performance and behaviour of black, urban elementary school children.

Nutritional status, school performance and behaviour were assessed in a group of black, inner-city school children. Thirty-five per cent of the children were obese by triceps skinfold thickness criteria. The primary care taker's responses to the Child Behavior Checklist (CBCL) and the hyperactivity subscale of the Conners' Parent's Questionnaire indicated that the obese children were more likely to have abnormal scores. On the CBCL subscale scores, obese girls had a significantly higher 'sex problems' score. Although the other subscale scores were not significantly different, there was a significant trend for obese boys and girls to score higher on the CBCL subscale scores. In addition, the proportion of obese children placed in special education or remedial class settings was twice that for non-obese children. These results add to the limited information available concerning obese black children, and are consistent with previous findings suggesting subtle behaviour differences in obese children.

Selection for kanamycin resistance in transformed petunia cells leads to the co-amplification of a linked gene.

A cell suspension culture was established from a transgenic petunia (Petunia hybrida L.) plant which carried genes encoding neomycin phosphotransferase II (nptII) and beta-glucuronidase (uidA, GUS). Two selection experiments were performed to obtain cell lines with increased resistance to kanamycin. In the first, two independently selected cell lines grown in the presence of 350 micrograms/ml kanamycin were eight to ten-fold more resistant to kanamycin than unselected cells. Increased resistance was correlated with amplification of the nptII gene and an increase in nptII mRNA levels. Selection for kanamycin resistance also produced amplification of the linked GUS gene, resulting in increased GUS mRNA levels and enzyme activity. Selected cells grown in the absence of kanamycin for twelve growth cycles maintained increased copy numbers of both genes, and GUS enzyme activity was also stably overexpressed. In a second selection experiment, a cell line grown continuously in medium containing 100 micrograms/ml kanamycin exhibited higher nptII and GUS gene copy numbers and an increase in GUS enzyme activity after eleven growth cycles. In this cell line, amplification of the two genes was accompanied by DNA rearrangement.

Differential activation and classification of cutaneous afferents in the rat.

1. A total of 312 cutaneous afferent units identified in the rat foot as belonging to one of nine major types of sensory receptors were included in the present study. A natural stimulus set was defined to differentiate optimally among those receptor types according to the distinguishing response patterns that it produced. It included air puffs, 30- and 300-Hz sinusoids, 200-mN force indentation of the skin, 1.2- and 6-N compressions of a skin fold, cooling the skin by 5 and 20 degrees C, warming by 5 degrees C, and heating by 15 degrees C. 2. The responses to predefined stimuli of 188 units were subjected to multivariate statistical analyses. The responses of an individual unit were measured as the number of impulses evoked by 10 stimuli, each lasting 10 s. Additionally, the number of impulses occurring for 5 s after withdrawal of a 200-mN indentation (1 of the 10 stimuli) was counted. 3. In discriminant analysis, the 11 stimulus variables predicted fairly correctly the grouping of afferent units into nine predetermined receptor categories (175 of 188, 93.1%), indicating a powerful ability to discriminate among different receptor types. Using hierarchical cluster analysis, afferent unit data described by 11 variables were divided into clusters that well represented prior receptor categories (170 of 188, 90.4%), suggesting the reliable application of this procedure to the classification of newly recorded cutaneous sensory receptors. 4. Eleven variables were then reduced to 7 on the basis of the results of factor analysis (95% of variance accounted for). The seven variables corresponded to 1.2-N compression, heating the skin by 15 degrees C, cooling the skin by 20 degrees C, 30- and 300-Hz sinusoids, withdrawal of a 200-mN indentation, and air puffs. 5. The seven selected variables correctly assigned afferent units into five modality-based categories in the discriminant solution (177 of 188, 94.1%). In the cluster solution, afferent units described by the seven selected variables were divided into clusters, most of whose members were modality specific (176 of 188, 93.6%). 6. The results indicate that cutaneous receptors can be divided into modality-specific groups according to similarities in their responses to seven stimulus variables. It is proposed that the stimulus set developed here and multivariate statistical methods can be used as powerful tools for the functional classification of central somatosensory neurons.