Evaluation of Attenuated Tumor Antigens and the Implications for Peptide-Based Cancer Vaccine Development.

INTRODUCTION: Peptide vaccines offer anti-tumor efficacy with very low toxicity. However, repeat stimulation with an immunogenic peptide leads to activation induced cell death (AICD), decreasing efficacy. We engineered variants of an immunogenic peptide (E39) and tested their ability to induce a robust, sustainable immune response. METHODS: Multiple variants of E39 were created by exchanging 1 or 2 amino acids. We tested the PBMC proliferation, cytokine production and cytolytic activity induced by each variant peptide. RESULTS: Repeated stimulation with E39 likely led to in vitro AICD, while stimulation with E39' led to T-cell proliferation with less evidence of AICD, modest cytokine production and high CTL activity. CONCLUSIONS: E39' appears to be the optimal variant of E39 for inducing effective long-term immunity.

MR1-restricted mucosal-associated invariant T (MAIT) cells respond to mycobacterial vaccination and infection in nonhuman primates.

Studies on mucosal-associated invariant T cells (MAITs) in nonhuman primates (NHP), a physiologically relevant model of human immunity, are handicapped due to a lack of macaque MAIT-specific reagents. Here we show that while MR1 ligand-contact residues are conserved between human and multiple NHP species, three T-cell receptor contact-residue mutations in NHP MR1 diminish binding of human MR1 tetramers to macaque MAITs. Construction of naturally loaded macaque MR1 tetramers facilitated identification and characterization of macaque MR1-binding ligands and MAITs, both of which mirrored their human counterparts. Using the macaque MR1 tetramer we show that NHP MAITs activated in vivo in response to both Bacillus Calmette-Guerin vaccination and Mycobacterium tuberculosis infection. These results demonstrate that NHP and human MR1 and MAITs function analogously, and establish a preclinical animal model to test MAIT-targeted vaccines and therapeutics for human infectious and autoimmune disease.

Differences between follicular waves 1 and 2 in patterns of emergence of 2-mm follicles, associated FSH surges, and ovarian vascular perfusion in heifers.

The emergence (first detection) of 2-mm follicles, FSH surges, and ovarian vascular perfusion for follicular wave 1 and surge 1 (n = 26) and wave 2 and surge 2 (n = 25) were studied daily in heifers. The day the future dominant follicle was closest to 5.5 mm was designated Day 0 for each wave. In wave 1, many 2-mm follicles (41%) emerged on Days -5 to -3, whereas FSH surge 1 did not begin until Day -3. Concentration of FSH increased abruptly in 1 day to a peak on the day of maximal number of emerging 2-mm follicles, although the day of maximal number relative to Day 0 differed among individuals. The first emergence of 2-mm follicles in wave 2 occurred concurrently with the first increase in the FSH of surge 2. In wave 1, ovarian resistance to vascular perfusion was negatively correlated (r = -0.48, P < 0.05) with a number of 2-mm follicles on Days -4 to -1 for ovaries that did not contain the preovulatory follicle; vascular perfusion increased with an increase in the number of small follicles. The following hypotheses were supported for wave 1 but not for wave 2: (1) an increase in the number of emerging 2-mm follicles of a follicular wave occurs before the beginning of an increase in FSH, (2) the day of maximal number of emerging 2-mm follicles occurs concurrently with an abrupt FSH increase on different days among individuals, and (3) the association between the number of emerging 2-mm follicles and the extent of ovarian vascular perfusion is positive.

Relationships among nitric oxide metabolites and pulses of a PGF2α metabolite during and after luteolysis in mares.

Hourly circulating concentrations of a PGF2α metabolite (PGFM), progesterone (P4), and LH were obtained from a reported project, and concentrations of nitric oxide (NO) metabolites (NOMs; nitrates and nitrites) were determined in eight mares. Unlike the reported project, hormone concentrations were normalized to the peak of the first PGFM pulse of luteolysis (early luteolysis), second PGFM pulse (late luteolysis), and a pulse after luteolysis. The duration of luteolysis was 23.1 ± 1.0 hours, and the peak of the first and second PGFM pulses occurred 6.5 ± 0.9 and 14.8 ± 0.8 hours after the beginning of luteolysis. Concentration of P4 decreased progressively within and between the PGFM pulses Changes were not detected in LH concentration in association with the PGFM pulses. Concentration of NOMs was greater (P < 0.05) at the peak of the PGFM pulse during early luteolysis (88.8 ± 15.0 µg/mL) than during late luteolysis (58.8 ± 9.0 µg/mL). Concentration of NOMs began to decrease (P < 0.05) 4 hours before the peak of the PGFM pulse of early luteolysis. Concentration began to increase (P < 0.05) an hour after the peak of the PGFM pulse of late luteolysis. An NOM decrease and increase was not detected during the PGFM pulse after luteolysis. On a temporal basis, results indicated that NO either is not required for luteolysis in mares or has a role in or responds only during late luteolysis. A caveat is that the relative contribution of the CL versus other body tissues to circulating concentrations of NOMs in mares has not been determined.

Spontaneous and experimental conversion of a regressing subordinate follicle of wave 1 to the dominant follicle of wave 2 in heifers.

Examination of daily ultrasound records from a previous study indicated that spontaneous conversion of a regressing largest subordinate follicle (SF) of wave 1 (SF1) to the dominant follicle (DF) of wave 2 (DF2) occurred on Day 6 or 7 (Day 0 = ovulation) in two of 28 heifers (7%). A conversion was considered definitive on the basis of no other SFs in the same ovary as SF1, thereby avoiding error in maintaining follicle identity. Spontaneous conversion appeared to involve an FSH fluctuation. In a separate study, experimental conversion of SF1 to DF2 was studied by ultrasonic imaging every 6 hours after ablating follicles other than SF1 when DF of wave 1 was close to 11.0 mm (hour 0). Diameter of SF1 decreased (P < 0.01) between hours -6 (7.8 ± 0.3 mm) and 0 (7.6 ± 0.3 mm). A decrease of 0.1 to 0.8 mm occurred in each heifer, indicating that SF1 was in early regression at hour 0. Conversion occurred in four of 12 (33%) heifers. A diameter increase (P < 0.05) in DF2 after conversion from SF1 occurred between hours 6 and 12. An increase (P < 0.05) in FSH occurred by hour 12 with and without conversion of SF1. Concentration of FSH at each of hours 30 to 48 was greater (P < 0.05) for nonconversion than that for conversion of SF1 to DF2 and greater (P < 0.05) for conversion than that for the basal concentration in controls (n = 7). The hypothesis that a regressing SF1 can be converted to DF2 by ablating other follicles was supported.

Circulating nitric oxide metabolites during luteolysis and the effect of luteinizing hormone on circulating nitric oxide metabolites in heifers.

Temporal relationships among circulating concentrations of nitric oxide metabolites (NOM), progesterone (P4), and luteinizing hormone (LH) within the hours of a PGFM pulse were studied during luteolysis in heifers. The peak of a PGFM pulse was designated Hour 0. All of the following increases and decreases were significant. Within a spontaneous PGFM pulse (experiment 1; n = 7), concentrations of P4 and LH decreased between Hours -1 and 0 and increased between Hours 0 and 1; NOM increased between Hours -1 and 2. In experiment 2, PGFM pulses were simulated by intrauterine infusion of PGF2α (PGF group, n = 6), and another group was also treated with acyline to inhibit LH secretion (acyline-PGF group, n = 6). Averaged over the two groups, concentration of P4 decreased between Hours -2 and 0, increased (rebounded) between Hours 0 and 1, and decreased after Hour 2. In the PGF group, concentration of LH decreased between Hours -2 and -0.5 and increased between Hour 0 and Hour 1.5 to a maximum at Hour 1.5; NOM decreased between Hours -2 and -1.5 and increased between Hours 0 and 1.5. In the acyline-PGF group, the effect of hour was not significant for concentrations of LH and NOM. The absence of an increase in NOM concentration when LH was inhibited is a novel finding. The hypotheses were supported that concentrations of LH and NOM are temporally related, and LH has a role in the increase in NOM within the hours of a PGFM pulse.

Horse species symposium: a novel approach to monitoring pathogen progression during uterine and placental infection in the mare using bioluminescence imaging technology and lux-modified bacteria.

Uterine and placental infections are the leading cause of abortion, stillbirth, and preterm delivery in the mare. Whereas uterine and placental infections in women have been studied extensively, a comprehensive examination of the pathogenic processes leading to this unsatisfactory pregnancy outcome in the mare has yet to be completed. Most information in the literature relating to late-term pregnancy loss in mares is based on retrospective studies of clinical cases submitted for necropsy. Here we report the development and application of a novel approach, whereby transgenically modified bacteria transformed with lux genes of Xenorhabdus luminescens or Photorhabdus luminescens origin and biophotonic imaging are utilized to better understand pathogen-induced preterm birth in late-term pregnant mares. This technology uses highly sensitive bioluminescence imaging camera systems to localize and monitor pathogen progression during tissue invasion by measuring the bioluminescent signatures emitted by the lux-modified pathogens. This method has an important advantage in that it allows for the potential tracking of pathogens in vivo in real time and over time, which was hitherto impossible. Although the application of this technology in domestic animals is in its infancy, investigators were successful in identifying the fetal lungs, sinuses, nares, urinary, and gastrointestinal systems as primary tissues for pathogen invasion after experimental infection of pregnant mares with lux-modified Escherichia coli. It is important that pathogens were not detected in other vital organs, such as the liver, brain, and cardiac system. Such precision in localizing sites of pathogen invasion provides potential application for this novel approach in the development of more targeted therapeutic interventions for pathogen-related diseases in the equine and other domestic species.

Effects of cannabis on lung function: a population-based cohort study.

The effects of cannabis on lung function remain unclear and may be different from those of tobacco. We compared the associations between use of these substances and lung function in a population-based cohort (n = 1,037). Cannabis and tobacco use were reported at ages 18, 21, 26 and 32 yrs. Spirometry, plethysmography and carbon monoxide transfer factor were measured at 32 yrs. Associations between lung function and exposure to each substance were adjusted for exposure to the other substance. Cumulative cannabis use was associated with higher forced vital capacity, total lung capacity, functional residual capacity and residual volume. Cannabis was also associated with higher airway resistance but not with forced expiratory volume in 1 s, forced expiratory ratio or transfer factor. These findings were similar among those who did not smoke tobacco. In contrast, tobacco use was associated with lower forced expiratory volume in 1 s, lower forced expiratory ratio, lower transfer factor and higher static lung volumes, but not with airway resistance. Cannabis appears to have different effects on lung function from those of tobacco. Cannabis use was associated with higher lung volumes, suggesting hyperinflation and increased large-airways resistance, but there was little evidence for airflow obstruction or impairment of gas transfer.

Associations between birth weight, early childhood weight gain and adult lung function.

BACKGROUND: Low birth weight is associated with lower values for spirometry in adults but it is not known if birth weight influences other measures of pulmonary function. It is also unclear whether postnatal growth affects adult lung function. The associations between birth weight, postnatal growth and adult lung function were assessed in an unselected birth cohort of 1037 children. METHODS: Birth weight, weight gain between birth and age 3 years, and lung function at age 32 years were measured. Analyses were adjusted for adult height and sex and further adjusted for multiple other potential confounding factors. RESULTS: Birth weight was positively correlated with spirometric (forced expiratory volume in 1 s and forced vital capacity) and plethysmographic (total lung capacity and functional residual capacity) lung function and with lung diffusing capacity. These associations persisted after adjustment for confounding factors including adult weight, exposure to cigarette smoke in utero and during childhood, personal smoking, socioeconomic status, asthma and gestational age. Weight gain between birth and age 3 years was also positively associated with lung diffusing capacity, and with higher values of lung volumes in men after adjustment for covariates. Neither birth weight nor postnatal weight gain was associated with airflow obstruction. CONCLUSIONS: Low birth weight and lower weight gain in early childhood are associated with modest reductions in adult lung function across a broad range of measures of lung volumes and with lower diffusing capacity. These findings are independent of a number of potential confounding factors and support the hypothesis that fetal and infant growth is a determinant of adult lung function.

Relationship between socioeconomic status and asthma: a longitudinal cohort study.

BACKGROUND: There is conflicting information about the relationship between asthma and socioeconomic status, with different studies reporting no, positive, or inverse associations. Most of these studies have been cross sectional in design and have relied on subjective markers of asthma such as symptoms of wheeze. Many have been unable to control adequately for potential confounding factors. METHODS: We report a prospective cohort study of approximately 1000 individuals born in Dunedin, New Zealand in 1972-3. This sample has been assessed regularly throughout childhood and into adulthood, with detailed information collected on asthma symptoms, lung function, airway responsiveness, and atopy. The prevalence of these in relation to measures of socioeconomic status were analysed with and without controls for potential confounding influences including parental history of asthma, smoking, breast feeding, and birth order using cross sectional time series models. RESULTS: No consistent association was found between childhood or adult socioeconomic status and asthma prevalence, lung function, or airway responsiveness at any age. Having asthma made no difference to educational attainment or socioeconomic status by age 26. There were trends to increased atopy in children from higher socioeconomic status families consistent with previous reports. CONCLUSIONS: Socioeconomic status in childhood had no significant impact on the prevalence of asthma in this New Zealand born cohort. Generalisation of these results to other societies should be done with caution, but our results suggest that the previously reported associations may be due to confounding.

The NCI/CIT microArray database (mAdb) system - bioinformatics for the management and analysis of Affymetrix and spotted gene expression microarrays.

A scalable, modular, enterprise-level system for both microarray databasing and analysis over the Internet has been developed over the past four years by the National Cancer Institute's Center for Cancer Research in collaboration with NIH's Center for Information Technology. This completely Web-based system, called mAdb (for microArray database), is currently supporting over 810 registered users and collaborators at NIH and contains over 22,000 microarray experiments, making it one of the largest collections of microarray data in existence. In addition, the mAdb system has been ported for the Netherlands Cancer Institute, the Genome Institute of Singapore, and the CDC. This system has been used for a wide variety of scientific experiments spanning the range from cancer to studies of early development, and for human, mouse, rat, yeast, and numerous microbial organisms.

Patterns of adherence to diabetes vision care guidelines: baseline findings from the Diabetic Retinopathy Awareness Program.

OBJECTIVES: (1) To describe baseline patterns of adherence to American Diabetes Association and American Academy of Ophthalmology vision care guidelines for diabetes in the Diabetic Retinopathy Awareness Program, and (2) to evaluate factors associated with nonadherence. This paper describes the baseline characteristics of a population enrolled in a prospective, randomized clinical trial. DESIGN: Cross-sectional study. PARTICIPANTS: Between October 1993 and May 1994, the study identified 2308 persons with diabetes, 18 years of age or older, who were residents of Suffolk County, New York, via a multimedia community-wide recruitment campaign. INTERVENTION AND METHODS: Eligibility for the trial was determined during a 20-minute phone interview, which included questions about vision care practices; diabetes management; and knowledge, attitudes, and beliefs about diabetes, vision, and diabetic retinopathy. This paper describes these patient characteristics at baseline. Eligible patients would be randomized subsequently to a 2-year diabetes educational intervention arm, which included mailed packets and newsletters focused on vision care, or to a control nonintervention arm. MAIN OUTCOME MEASURE: Nonadherence to guidelines at baseline was defined as the absence of a dilated eye examination during the year before recruitment into the study. RESULTS: Of the 2308 persons interviewed, 813 (35%) did not follow the vision care guidelines; two thirds of this group reported no eye examination in the year before the interview, and one third had an undilated examination. Ophthalmologists performed 49% of the examinations in the nonadherent group, versus 86% in the adherent group. In logistic regression analyses, factors related to nonadherence were: younger age (odds ratio [OR] = 0.97), type 2 diabetes with or without insulin use (OR = 1.62 and 1.99, respectively), shorter diabetes duration (OR = 0.97), last eye examination performed by an optometrist (OR = 5.32) or other nonophthalmologist (OR = 4.29), less practical knowledge about diabetes (OR = 1.57), and no prior formal diabetes education (OR = 1.30). CONCLUSIONS: Within this population, more than one third of participants had not been following vision care guidelines. Nonadherence was linked to several potentially modifiable factors; changes in these factors could enhance the early detection of diabetic retinopathy.

S1 analysis of messenger RNA using single-stranded DNA probes.

This method takes advantage of the ability of oligonucleotides to be efficiently labeled to a high specific activity at the 5' end through the use of kinase. The oligonucleotide is hybridized to a specific single-stranded template containing the complementary sequence to the oligonucleotide, and this hybrid is extended through the use of the Klenow fragment of E. coli DNA polymerase I. The mixture is cut with a restriction enzyme to give the probe a defined 3' end, and the probe is isolated on an alkaline agarose gel. Before using this protocol it is first helpful to have an M13 clone. If this is unavailable, a double-stranded plasmid clone of the region to be studied may be used, as described in an alternate protocol. Another alternate protocol describes the use of long oligonucleotides as probes for S1 analysis (useful for rapid and easy quantitation of the level of mRNA produced from a characterized promoter). For the mapping of the 5' end of an RNA species, hybridization of the probe to RNA is then carried out. S1 nuclease is added to digest all of the unhybridized portion of the probe. Electrophoresis of the hybrid on a denaturing polyacrylamide gel allows a determination of the length of the remaining DNA fragment. This length equals the distance between the 5' end of the probe to the 5' end of the RNA, defining the transcriptional start site to the nucleotide. By performing the hybridization reaction in vast probe excess, quantitation of the relative amounts of RNA can be estimated between samples.

Linker-scanning mutagenesis of DNA.

Two protocols are described in which clusters of point mutations are introduced throughout a sequence of interest that has been cloned into a plasmid vector. The first protocol uses complementary oligonucleotides and requires a unique restriction site adjacent to the region that is to be mutagenized. A nested series of deletion mutations is first generated in the region. A pair of complementary oligonucleotides are synthesized to fill in the gap in the sequence of interest between the linker at the deletion endpoint and the nearby restriction site. The linker sequence actually provides the desired clusters of point mutations as it is moved or "scanned" across the region by its position at the varied endpoints of the deletion mutation series. An Alternate Protocol makes use of site-directed mutagenesis procedures to introduce smaller clusters of point mutations throughout the target region.

Recruiting participants for community-based research: the Diabetic Retinopathy Awareness Program.

PURPOSE: Recruiting participants is a major challenge for population studies. We present the recruitment methods followed by the Diabetic Retinopathy Awareness Program (DRAP), a community-based, randomized, masked, controlled trial to meet and exceed its sample size goals. METHODS: A county-wide multi-media promotional campaign to recruit and enroll participants in the trial was planned and executed from October 1993 through April 1994, with the assistance of the local news media and community and professional groups. A toll-free 800 number recruitment line was established, and postage-paid recruitment postcards distributed. The trial was designed to examine whether a mailed educational intervention could increase compliance with vision care guidelines among persons with diabetes in the community. RESULTS: A total of 2308 persons with diabetes were interviewed for eligibility and 813 enrolled in the intervention trial, exceeding the original recruitment goals of 1800 and 600, respectively. Those who completed the enrollment interview reflected county demographics. During recruitment, newspaper, television and radio stories featured the study; pharmacies and physician offices displayed study materials; public service announcements appeared in local print and broadcast media. The largest single recruitment response was a local television news report, followed by a newspaper story. CONCLUSIONS: These experiences substantiate the need for a comprehensive coordinated approach, using planned multiple sources, to achieve recruitment success. By engaging the lay and professional communities along with the media, recruitment costs can be kept to a minimum. Participant costs can be minimized by employing a toll-free number and eliminating study participant travel, thus allowing for inclusion of traditionally underserved populations. This approach is applicable to other studies, where community-based participation is desired.

Pre-B acute lymphoblastic leukemia with b3a2 (p210) and e1a2 (p190) BCR-ABL fusion transcripts relapsing as chronic myelogenous leukemia with a less differentiated b3a2 (p210) clone.

The Philadelphia chromosome translocation t(9;22)(q34;q11) may give rise to different BCR/ABL fusion mRNAs due to different genomic breakpoints and alternative splicing. The e1a2, b2a2 or b3a2 and c3a2 fusion mRNAs encode distinct fusion proteins (p190, p210 and p230, respectively), which are associated with different forms of leukemogenesis in humans and animal models. Our patient presented with acute pre-B cell lymphoblastic leukemia (ALL) with normal cytogenetics. After 3 years of standard ALL therapy, he relapsed with t(9;22)-positive chronic myelogenous leukemia (CML). Retrospective molecular analyses of the pre-treatment pre-B cell ALL sample showed the b3a2 (p210) and e1a2 (p190) BCR/ABL fusion transcripts. Only the b3a2 (p210) transcript was detected at relapse. Southern and immunoglobulin heavy chain (IgH) analyses of the presentation and relapse samples revealed an identical BCR rearrangement in both samples. However, only the ALL sample harbored an IgH gene rearrangement. These findings show a clonal relationship between the more differentiated pre-B cell and less differentiated CML clones and that the p210 and p190 fusion mRNAs were alternatively spliced from a single genomic breakpoint. Our patient's unusual molecular findings provide circumstantial evidence that the p190 protein may promote a more differentiated phenotype in a comparatively less differentiated p210-transformed precursor cell.

Prevalence and correlates of survival sex among runaway and homeless youth.

OBJECTIVE: This study examined the prevalence and correlates of survival sex among runaway and homeless youths. METHODS: A nationally representative sample of shelter youths and a multicity sample of street youths were interviewed. RESULTS: Approximately 28% of street youths and 10% of shelter youths reported having participated in survival sex, which was associated with age, days away from home, victimization, criminal behaviors, substance use, suicide attempts, sexually transmitted disease, and pregnancy. CONCLUSIONS: Intensive and ongoing services are needed to provide resources and residential assistance to enable runaway and homeless youths to avoid survival sex, which is associated with many problem behaviors.

Pregnancy among three national samples of runaway and homeless youth.

PURPOSE: To compare estimates of the prevalence of pregnancy among runaway and homeless youth between the ages of 14 and 17 years in various settings with each other and with youth in the general population. METHODS: Comparisons used three surveys of youth: (a) the first nationally representative survey of runaway and homeless youth residing in federally and nonfederally funded shelters, (b) a multicity survey of street youth, and (c) a nationally representative household survey of youth with and without recent runaway and homeless experiences. RESULTS: Youth living on the streets had the highest lifetime rates of pregnancy (48%), followed by youth residing in shelters (33%) and household youth (<10%). CONCLUSIONS: Shelter and street youth were at much greater risk of having ever been pregnant than were youth in households, regardless of whether they had recent runaway or homeless experiences. Such youth need comprehensive services, including pregnancy prevention, family planning, and prenatal and parenting services.

PIP: This study estimated and compared adolescent pregnancy rates among runaway and homeless adolescents in several settings. Data were obtained through 3 surveys: 1) a 1992 Youth Risk Behavior Survey (YRBS) among a nationally representative sample with recent runaway and homeless youth experiences; 2) the first nationally representative survey among a multistage sample of runaway and homeless youth living in federally and nonfederally funded youth shelters; and 3) the first multi-city survey of street youth. Data were collected among youth aged 14-17 years in 1992. Findings indicate that in all surveys youth were usually pregnant only once, but many street and shelter youth had 2 or more pregnancies. About 10% of both street and shelter youth reported being pregnant at the time of the interview. Significant differences occurred by age among street youth and for length of time away among both street and shelter youth. About 50% of street youth had a pregnancy experience compared to about 33% living in shelters. Less than 10% of household runaway youth had a pregnancy experience. Differences in rates were not statistically significant. The findings are based on self-reports, a purposive street sample, and potentially duplicate reporting among street youth. The high pregnancy rates are attributed, in part, to the prevalence of sexual abuse and other related reasons. 21-60% reported sexual abuse, compared to only 3-12% among the general population. A strong need exists for preventive/prenatal services for runaway and street youth.

The prevalence of homelessness among adolescents in the United States.

OBJECTIVES: Homeless adolescents represent one of the nation's most vulnerable populations. This study reports the 12-month prevalence of homeless episodes among US adolescents. METHODS: Personal, audiotaped interviews were conducted in 1992 and 1993 with a representative household sample of 6496 adolescents aged 12 to 17 as part of the Youth Risk behavior Survey sponsored by the Centers for Disease Control and Prevention. Respondents reported whether they had spent the night in any of a variety of locations other than home during the previous 12 months. RESULTS: Altogether, 7.6% of the youths questioned reported that they had spent at least 1 night in youth or adult shelter (3.3%), public place (2.2%), an abandoned building (1.0%), outside 2.2%), underground (0.4%), or with a stranger (1.1%). Boys were much more likely than girls to report having experienced a homeless episode. CONCLUSIONS: This study suggests that homelessness among adolescents is not simply an urban problem and that prevention programs targeting homeless youths should be implemented nationwide. Additional research is needed to assess the frequency and duration of homeless experiences. Future studies of homelessness in the general population should include questions pertinent to adolescents.

Human geranylgeranyl diphosphate synthase: isolation of the cDNA, chromosomal mapping and tissue expression.

We report the nucleotide sequence of human geranylgeranyl diphosphate (GGPP) synthase cDNA isolated from a fetal heart library. The 2.5 kb cDNA encodes a protein of 34 kDa. The protein contains six domains that have been identified previously in many other prenyltransferases. Recombinant, purified histidine-tagged protein exhibited the enzymatic properties associated with GGPP synthase, namely the synthesis of GGPP from farnesyl diphosphate and isopentenyl diphosphate. Transient transfection of mammalian cells with a plasmid encoding the putative GGPP synthase resulted in a 55-fold increase in GGPP synthase activity. Taken together, these results establish that the cDNA encodes the mammalian GGPP synthase protein. The mRNA for GGPP synthase was expressed ubiquitously. Of the 16 human tissues examined, the highest expression of the mRNA was in testis. The mRNA levels in cultured HeLa cells were unaffected by alterations in cellular sterol levels and contrasted with the significant regulation of isopentenyl diphosphate synthase mRNA under these same conditions. Fluorescent in situ hybridization was used to map the single gene encoding human GGPP synthase to chromosome 1q43.