Biomedical subjects
J M Roberts
Publications and source records attributed to J M Roberts.
A syndrome of multiorgan hyperplasia with features of gigantism, tumorigenesis, and female sterility in p27(Kip1)-deficient mice.
SUMMARY: Targeted disruption of the murine p27(Kip1) gene caused a gene dose-dependent increase in animal size without other gross morphologic abnormalities. All tissues were enlarged and contained more cells, although endocrine abnormalities were not evident. Thymic hyperplasia was associated with increased T lymphocyte proliferation, and T cells showed enhanced IL-2 responsiveness in vitro. Thus, p27 deficiency may cause a cell-autonomous defect resulting in enhanced proliferation in response to mitogens. In the spleen, the absence of p27 selectively enhanced proliferation of hematopoietic progenitor cells. p27 deletion, like deletion of the Rb gene, uniquely caused neoplastic growth of the pituitary pars intermedia, suggesting that p27 and Rb function in the same regulatory pathway. The absence of p27 also caused an ovulatory defect and female sterility. Maturation of secondary ovarian follicles into corpora lutea, which express high levels of p27, was markedly impaired.
Requirement of p27Kip1 for restriction point control of the fibroblast cell cycle.
Cells deprived of serum mitogens will either undergo immediate cell cycle arrest or complete mitosis and arrest in the next cell cycle. The transition from mitogen dependence to mitogen independence occurs in the mid-to late G1 phase of the cell cycle and is called the restriction point. Murine Balb/c-3T3 fibroblasts deprived of serum mitogens accumulated the cyclin-dependent kinase (CDK) inhibitor p27Kip1. This was correlated with inactivation of essential G1 cyclin-CDK complexes and with cell cycle arrest in G1. The ability of specific mitogens to allow transit through the restriction point paralleled their ability to down-regulate p27, and antisense inhibition of p27 expression prevented cell cycle arrest in response to mitogen depletion. Therefore, p27 is an essential component of the pathway that connects mitogenic signals to the cell cycle at the restriction point.
The yeast CDC16 and CDC27 genes restrict DNA replication to once per cell cycle.
CDC16 and CDC27 were identified as genes in S. cerevisiae necessary to limit DNA replication to once per cell cycle. A screen for mutants that overreplicated their DNA uncovered new conditional alleles that cause accumulation of up to 8C DNA. DNA overreplication involves all chromosomes and does not require passage through mitosis or another START. It occurs within a single cell cycle and can cause arrest at the MEC1 checkpoint. Remarkably, Clb2-Cdc28 activity remains elevated in the overreplicating cells. These observations distinguish CDC16 and CDC27 from other mutants that accumulate extra DNA after completing an aberrent mitosis, or skipping mitosis altogether, and entering a second, inappropriate G1 and S phase. CDC16 and CDC27 may contribute to replication control by targeted proteolysis of an S phase initiator.
Regulation of cyclin E transcription by E2Fs and retinoblastoma protein.
Cyclin E is critical for the advance of cells through the G1 phase of their growth cycle. Transcription of the cyclin E gene is known to be cell cycle-dependent. We have shown previously that mRNA levels of cyclin E are regulated positively by mitogens and negatively by TGF-beta. Much circumstantial evidence implicates both E2F transcription factors and the retinoblastoma protein (pRB) in the control of cyclin E expression. However, the molecular basis of this control has remained unclear. We report here the cloning of the cyclin E promoter and the identification of several putative E2F binding sites within the promoter sequence. We have found that cell cycle regulation of cyclin E transcription is mediated by E2F binding sites present in the promoter. The activity of this promoter can be regulated negatively by pRB. Our results suggest the operation of a positive-feedback loop in late G1 that functions to ensure continued cyclin E expression and pRB inactivation.
The length of the cervix and the risk of spontaneous premature delivery. National Institute of Child Health and Human Development Maternal Fetal Medicine Unit Network.
BACKGROUND: The role of the cervix in the pathogenesis of premature delivery is controversial. In a prospective, multicenter study of pregnant women, we used vaginal ultrasonography to measure the length of the cervix; we also documented the incidence of spontaneous delivery before 35 weeks' gestation. METHODS: At 10 university-affiliated prenatal clinics, we performed vaginal ultrasonography at approximately 24 and 28 weeks of gestation in women with singleton pregnancies. We then assessed the relation between the length of the cervix and the risk of spontaneous preterm delivery. RESULTS: We examined 2915 women at approximately 24 weeks of gestation and 2531 of these women again at approximately 28 weeks. Spontaneous preterm delivery (at less than 35 weeks) occurred in 126 of the women (4.3 percent) examined at 24 weeks. The length of the cervix was normally distributed at 24 and 28 weeks (mean [+/- SD], 35.2 +/- 8.3 mm and 33.7 +/- 8.5 mm, respectively). The relative risk of preterm delivery increased as the length of the cervix decreased. When women with shorter cervixes at 24 weeks were compared with women with values above the 75th percentile, the relative risks of preterm delivery among the women with shorter cervixes were as follows: 1.98 for cervical lengths at or below the 75th percentile (40 mm), 2.35 for lengths at or below the 50th percentile (35 mm), 3.79 for lengths at or below the 25th percentile (30 mm), 6.19 for lengths at or below the 10th percentile (26 mm), 9.49 for lengths at or below the 5th percentile (22 mm), and 13.99 for lengths at or below the 1st percentile (13 mm) (P < 0.001 for values at or below the 50th percentile; P = 0.008 for values at or below the 75th percentile). For the lengths measured at 28 weeks, the corresponding relative risks were 2.80, 3.52, 5.39, 9.57, 13.88, and 24.94 (P < 0.001 for values at or below the 50th percentile; P = 0.003 for values at the 75th percentile). CONCLUSIONS: The risk of spontaneous preterm delivery is increased in women who are found to have a short cervix by vaginal ultrasonography during pregnancy.
Fasting serum triglycerides, free fatty acids, and malondialdehyde are increased in preeclampsia, are positively correlated, and decrease within 48 hours post partum.
OBJECTIVE: We tested the hypothesis that serum free (nonesterified) fatty acid and triglyceride concentrations are increased in nulliparous women with preeclampsia relative to women with uncomplicated pregnancies and that these lipids decrease post partum, consistent with the known resolution of clinical symptoms. The relationships between serum concentrations of these lipids and the lipid peroxidation metabolite malondialdehyde were also examined. STUDY DESIGN: Predelivery and 24 to 48 hour postpartum venous blood samples were collected from eight women with preeclampsia and nine women with uncomplicated pregnancies after an 8- to 10-hour fast. Sera were analyzed for concentrations of triglycerides, free fatty acids, total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, and malondialdehyde. RESULTS: Antepartum serum triglyceride and free fatty acid concentrations were increased approximately twofold in women with preeclampsia relative to uncomplicated pregnancies (p <0.02 and 0.004, respectively). Total, high-density lipoprotein, and low-density lipoprotein cholesterol concentrations did not differ between groups. Concentrations of all lipids decreased significantly in both groups within 48 hours post partum. However, triglyceride and free fatty acid concentrations remained higher in women with preeclampsia (p<0.006, both variables). Triglyceride and free fatty acid concentrations correlated positively, both ante partum (R2 0.42, p<0.01) and post partum (R2 0.39, p<0.02). Antepartum concentrations of malondialdehyde were 50% higher in women with preeclampsia (p<0.01) and decreased post partum (p <0.02) but did not decrease in controls (p = 0.07). Antepartum serum triglycerides and free fatty acids correlated positively with malondialdehyde concentrations (R2 0.38, p <0.02, both cases). CONCLUSION: Triglycerides and free fatty acids, but not cholesterol, are increased in preeclampsia and correlate with the lipid peroxidation metabolite malondialdehyde. We speculate that these interactions may contribute to endothelial cell dysfunction in preeclampsia.
Urine but not plasma nitric oxide metabolites are decreased in women with preeclampsia.
OBJECTIVE: Nitric oxide is a potent vasorelaxant produced by endothelial cells. We tested the hypothesis that urinary and perhaps plasma nitric oxide metabolites would be reduced in women with preeclampsia. STUDY DESIGN: Plasma and urine from 14 women meeting strict clinical criteria for the diagnosis of preeclampsia and 20 normal nulliparous women were assayed for the stable metabolites of nitric oxide, nitrate and nitrite. RESULT: There was no significant difference of plasma concentrations of nitrate and nitrite between women with preeclampsia and women with normal pregnancies (32.7 +/- 3.1 vs 25.8 +/- 2.4 micromol/L). Plasma creatinine levels were elevated in women with preeclampsia (0.85 +/- 0.09 vs 0.66 +/- 0.02 mg/dl, p<0.01), indicating a reduced glomerular filtration rate. Urine concentrations of nitrate and nitrite normalized by creatinine excretion were significantly lower in women with preeclampsia compared with normal pregnant women (0.37 +/- 0.06 vs 0.69 +/- 0.11 micromol of nitrite per milligram creatinine, p. <0.05). CONCLUSIONS: Our study using concomitant measurement of plasma and urine nitrate and nitrite suggests a reduced production of nitric oxide in women with preeclampsia compared with normal pregnant women.
Hyperuricemia and xanthine oxidase in preeclampsia, revisited.
Hyperuricemia is associated with the severity of preeclampsia and with fetal outcome. Traditionally the high uric acid concentration in preeclampsia has been attributed soley to renal dysfunction. Preeclampsia is also characterized by increased free radical formation and elevated oxidative stress. Xanthine dehydrogenase/oxidase produces uric acid. Xanthine dehydrogenase/oxidase is present as two isoforms in vivo. Uric acid production is coupled with formation of reactive oxygen species when the enzyme is in the oxidase form. Several factors can increase the holoenzyme activity and the conversion of xanthine dehydrogenase/oxidase to its oxidase form. These factors include hypoxia-reperfusion, cytokines, and increased substrate availability (xanthine and hypoxanthine). Preeclampsia is characterized by hyperuricemia and signs of increased formation of reactive oxygen species and decreased levels of antioxidants. Preeclampsia is also characterized by shallow implantation, producing a relatively hypoxic maternal-fetal interface, and increased turnover of trophoblast tissue, which can result in higher xanthine and hypoxanthine concentrations and higher levels of circulating cytokines. These mechanisms can lead to increased production of uric acid and free radicals and contribute to the hyperuricemia and increased oxidative stress present in preeclampsia.
Mechanical stress eliminates the effects of plasma from patients with preeclampsia on endothelial cells.
OBJECTIVE: Our purpose was to determine whether mechanical deformation alters in vitro effects of plasma from patients with preeclampsia on endothelial cell function to produce a paradigm similar to the in vivo disease state. STUDY DESIGN: The effects of 2% plasma from 12 patients with preeclampsia and 12 normal pregnant women on prostacyclin, nitric oxide, and endothelin production by cultured endothelial cells were measured in the presence or absence of cyclic stretch and laminar shear stress. RESULTS: In the absence of mechanical stress plasma from patients with preeclampsia resulted in greater prostacyclin and nitric oxide production (but no change in endothelin production) compared with plasma from normal pregnant women. Cyclic stretch did not affect prostacyclin or endothelin production but produced similar increases in nitric oxide production in cells exposed to plasma from the two groups. Shear stress markedly increased prostacyclin and nitric oxide production (but did not alter endothelin production). In the presence of shear stress there were no differences in production rates of nitric oxide or prostacyclin between cells exposed to plasma from the two groups. CONCLUSION: Shear stress alters the effects of plasma on endothelial cells. This is an important caveat in the interpretation of previous static in vitro culture studies and may partially explain the dichotomy between in vitro studies and in vivo findings in preeclampsia.
Adhesion-dependent cell cycle progression linked to the expression of cyclin D1, activation of cyclin E-cdk2, and phosphorylation of the retinoblastoma protein.
Growth factors and cell anchorage jointly regulate transit through G1 in almost all cell types, but the cell cycle basis for this combined requirement remains largely uncharacterized. We show here that cell adhesion and growth factors jointly regulate the cyclin D1- and E-dependent kinases. Adhesion to substratum regulates both the induction and translation of cyclin D1 mRNA. Nonadherent cells fail to phosphorylate the retinoblastoma protein (Rb), and enforced expression of cyclin D1 rescues Rb phosphorylation and entry into S phase when G1 cells are cultured in the absence of substratum. Nonadherent cells also fail to activate the cyclin E-associated kinase, and this effect can be linked to an increased association of the cdk inhibitors, p21 and p27. These data describe a striking convergence in the cell cycle controls used by the two major signal transduction systems responsible for normal and abnormal cell growth. Taken together with our previous studies showing adhesion-dependent expression of cyclin A, they also establish the cell cycle basis for explaining the combined requirement for growth factors and the extracellular matrix in transit through the Rb checkpoint, entry into S phase, and anchorage-dependent growth.
Natural immune response to the C-terminal 19-kilodalton domain of Plasmodium falciparum merozoite surface protein 1.
We have characterized the natural immune responses to the 19-kDa domain of merozoite surface protein 1 in individuals from an area of western Kenya in which malaria is holoendemic. We used the three known natural variant forms of the yeast-expressed recombinant 19-kDa fragment that are referred to as the E-KNG, Q-KNG, and E-TSR antigens. T-cell proliferative responses in individuals older than 15 years and the profile of immunoglobulin G (IgG) antibody isotypes in individuals from 2 to 74 years old were determined. Positive proliferative responses to the Q-KNG antigen were observed for 54% of the individuals, and 37 and 35% of the individuals responded to the E-KNG and E-TSR constructs, respectively. Considerable heterogeneity in the T-cell proliferative responses to these three variant antigens was observed in different individuals, suggesting that the 19-kDa antigen may contain variant-specific T epitopes. Among responses of the different isotypes of the IgG antibody, IgG1 and IgG3 isotype responses were predominant, and the prevalence and levels of the responses increased with age. We also found that a higher level of IgG1 antibody response correlated with lower parasite density among young age groups, suggesting that IgG1 antibody response may play a role in protection against malaria. However, there was no correlation between the IgG3 antibody level and protection. Furthermore, we observed that although the natural antibodies cross-reacted with all three variant 19-kDa antigens, IgG3 antibodies in 12 plasma samples recognized only the E-KNG and Q-KNG constructs and not the E-TSR antigen. This result suggests that the fine specificity of IgG3 antibodies differentiates among variant-specific natural B-cell determinants in the second epidermal growth factor domain (KNG and TSR) of the antigen.
Plasma of preeclamptic women stimulates and then inhibits endothelial prostacyclin.
We propose that the dichotomy between the in vivo reduction in intravascular prostacyclin production that occurs in preeclampsia and the in vitro stimulatory effect of plasma from preeclamptic patients on endothelial cell prostacyclin production is due to differential effects of chronic versus acute exposure to the plasma. We studied the acute versus chronic effects of 2% plasma from healthy pregnant and preeclamptic subjects by measuring endothelial prostacyclin production at different time periods after exposure to plasma. To determine whether such effects were specific to prostacyclin, we also measured prostaglandin E2 production. To determine whether chronic changes in prostacyclin production resulted from altered cellular responsiveness, we stimulated cells that had been exposed to plasma for 72 hours with arachidonic acid and measured prostaglandin production. Preliminary characterization of the plasma factor or factors responsible for alterations in prostaglandin production was performed. After 24 hours cells exposed to plasma from preeclamptic women produced more prostacyclin and prostaglandin E2 than cells exposed to plasma from healthy pregnant women. In contrast, after 72 hours exposure to plasma from preeclamptic women resulted in less endothelial cell prostacyclin production than exposure to plasma from healthy pregnant women, but there were no such differences in prostaglandin E2 production. Cells that had been exposed to plasma from preeclamptic women for 72 hours produced less prostacyclin but the same quantity of prostaglandin E2 after stimulation with arachidonic acid than cells exposed to plasma from healthy pregnant women. The plasma factor or factors responsible for altered prostacyclin production were sensitive to heat, acid, and proteases. In contrast to acute exposure, chronic exposure to plasma from preeclamptic women alters endothelial cells to result in decreased prostacyclin production, an observation consistent with in vivo findings.
The Santa Lucia strain of Plasmodium falciparum as a model for vaccine studies. I. Development in Aotus lemurinus griseimembra monkeys.
The Santa Lucia strain of Plasmodium falciparum and the Aotus lemurinus griseimembra monkey are proposed as models for the testing of sporozoite vaccines and transmission-blocking vaccines. Approximately 85% of splenectomized monkeys were infected when fed upon by 10 or more heavily infected Anopheles freeborni mosquitoes. Sporozoite-induced infections in monkeys with or without previous infection with P. vivax readily infected mosquitoes, thus making them candidates for testing transmission-blocking vaccines.
Malaria parasite infection during pregnancy and at delivery in mother, placenta, and newborn: efficacy of chloroquine and mefloquine in rural Malawi.
Despite international recommendations to use malaria treatment and prevention in pregnant women in malaria-endemic areas, few studies have evaluated the efficacy of available antimalarial regimens. This issue is of particular concern in the face of spreading chloroquine (CQ)-resistance of Plasmodium falciparum in malarious areas of sub-Saharan Africa. In a prospective trial in rural Malawian pregnant women, we examined three regimens using CQ (including the existing national policy regimen) and one regimen using mefloquine (MQ). The efficacy of the regimens was determined by comparing rates of clearance of initial parasitemia; prevention of breakthrough infection; and parasitemia at delivery in maternal peripheral blood, placental blood, and in infant umbilical cord blood. Among 1,528 parasitemic women at enrollment, 281 (18.4%) had persistent infections; and among 1,852 initially aparasitemic women, 320 (17.3%) had breakthrough parasitemia on one or more follow-up visits. Compared with women on MQ, women on a CQ regimen were at significantly greater risk of persistent and breakthrough infection (odds ratios [OR] = 30.9 and 11.1, respectively, P < 10(-6)). Other significant risk factors for persistent and breakthrough infections in a multivariate model included first pregnancy; enrollment in the rainy or postrainy season; maternal age < or = 25 years; seropositivity to the human immunodeficiency virus (HIV) (persistent infections only); and no use of antimalarial prophylaxis before enrollment (breakthrough infections only). At delivery, compared with women on MQ, women on a CQ regimen were at significantly greater risk of peripheral, placental, or umbilical cord blood parasitemia (OR = 8.7, 7.4, and 4.1, respectively, P < 10(-6)). Additional risk factors for parasitemia at delivery in multivariate models included first pregnancy; delivery in the rainy or postrainy season; HIV-seropositivity; and maternal age < or = 25 years (risk for peripheral and placental blood parasitemia only). Maternal anemia (hematocrit < 30%) at enrollment or at delivery was not associated with persistent or breakthrough parasitemia or parasitemia at deliver in these multivariate models. While factors leading to increased malaria parasite exposure (high transmission seasons) and lowered or altered host immune response (low pregnancy number, young age, and HIV infection) are important risk factors for malaria in pregnant women, the use of an ineffective intervention (CQ in a setting with CQ-resistant parasites) was the most important determinant of P. falciparum parasitemia in these pregnant women. Strategies to reduce the impact of malaria in pregnant women must use efficacious interventions and may need to consider targeting the intervention to the most susceptible women during the seasons of high malaria exposure.
Preeclampsia: not simply pregnancy-induced hypertension.
Once classified as a seizure disorder and later as a hypertensive condition, preeclampsia is actually a multisystem disease. It remains a leading cause of maternal and neonatal morbidity. Clinical challenges include identifying patients who are at risk of serious complications and deciding whether it is safer for the fetus to be delivered or to remain in the uterus.
Malaria surveillance--United States, 1992.
PROBLEM/CONDITION: Malaria is caused by one of four species of Plasmodium (i.e., P. falciparum, P. vivax, P. ovale, or P. malariae) and is transmitted by the bite of an infective female Anopheles sp. mosquito. Most malaria cases in the United States occur among persons who have traveled to areas that have ongoing transmission. However, cases are transmitted occasionally through exposure to infected blood products, by congenital transmission, or by local mosquito-borne transmission. Malaria surveillance is conducted to identify episodes of local transmission and to guide prevention recommendations. REPORTING PERIOD COVERED: Cases with onset of illness during 1992. DESCRIPTION OF SYSTEM: Malaria cases were identified at the local level (i.e., by healthcare providers or through laboratory-based surveillance). All suspected cases were confirmed by slide diagnosis and then reported to the respective state health department and to CDC. RESULTS: CDC received reports of 910 cases of malaria that had onset of symptoms during 1992 among persons in the United States and its territories. In comparison, 1,046 cases were reported for 1991, representing a decrease of 13% in 1992. P. vivax, P. falciparum, P. malariae, and P. ovale were identified in 51%, 33%, 4%, and 3% of cases, respectively. The species was not identified in the remaining 9% of cases. The number of reported malaria cases that had been acquired in Africa by U.S. civilians decreased 38%, primarily because the number of P. falciparum cases declined. Of U.S. civilians whose illnesses were diagnosed as malaria, 81% had not taken a chemoprophylactic regimen recommended by CDC. Seven patients had acquired their infections in the United States. Seven deaths were attributed to malaria. INTERPRETATION: The decrease in the number of P. falciparum cases in U.S. civilians could have resulted from a change in travel patterns, reporting errors, or increased use of more effective chemoprophylaxis regimens. ACTIONS TAKEN: Additional information was obtained concerning the seven fatal cases and the seven cases acquired in the United States. Malaria prevention guidelines were updated and disseminated to health-care providers. Persons traveling to a malaria-endemic area should take the recommended chemoprophylaxis regimen and use personal protection measures to prevent mosquito bites. Any person who has been to a malarious area and who subsequently develops a fever or influenza-like symptoms should seek medical care, which should include a blood smear for malaria. The disease can be fatal if not diagnosed and treated at an early stage of infection. Recommendations concerning prevention and treatment of malaria can be obtained from CDC.