The Anopheles gambiae adult midgut peritrophic matrix proteome.

Malaria is a devastating disease. For transmission to occur, Plasmodium, the causative agent of malaria, must complete a complex developmental cycle in its mosquito vector. Thus, the mosquito is a potential target for disease control. Plasmodium ookinetes, which develop within the mosquito midgut, must first cross the midgut's peritrophic matrix (PM), a thick extracellular sheath that completely surrounds the blood meal. The PM poses a partial, natural barrier against parasite invasion of the midgut and it is speculated that modifications to the PM may lead to a complete barrier to infection. However, such strategies require thorough characterization of the structure of the PM. Here, we describe for the first time, the complete PM proteome of the main malaria vector, Anopheles gambiae. Altogether, 209 proteins were identified by mass spectrometry. Among them were nine new chitin-binding peritrophic matrix proteins, expanding the list from three to twelve peritrophins. Lastly, we provide a model for the putative interactions among the proteins identified in this study.

Storage and secretion of Ag-Aper14, a novel peritrophic matrix protein, and Ag-Muc1 from the mosquito Anopheles gambiae.

The gene Ag-Aper14 encodes a novel peritrophic matrix (or peritrophic membrane; PM) protein in the mosquito Anopheles gambiae. The Ag-Aper14 protein is merely 89 amino acids long and has a single putative chitin-binding domain. Prior to blood feeding, the Ag-Aper14 protein is stored in secretory vesicles next to the epithelial cell lumenal surface. Immunoelectron microscopy has revealed that Ag-Aper14 co-localizes to the same secretory vesicles as another PM protein, Ag-Aper1, indicating a common mode of regulated secretion. Conversely, Ag-Muc1, an epithelial cell-surface protein, does not co-localize to these secretory vesicles and is detected only on the cell surface. After blood feeding, Ag-Aper14 is secreted and incorporated into the PM that surrounds the ingested blood.

Storage and secretion of the peritrophic matrix protein Ag-Aper1 and trypsin in the midgut of Anopheles gambiae.

The gene Ag-Aper1 encodes a peritrophic matrix (PM) protein from the mosquito Anopheles gambiae. Ag-Aper1 gene expression and protein localization in the mosquito midgut were studied during the course of a blood meal. Ag-Aper1 mRNA abundance does not change appreciably during the course of blood ingestion and digestion. Prior to a blood meal, the protein is stored in secretory vesicles of midgut epithelial cells. Moreover, Ag-Aper1 colocalizes to the same secretory vesicles as trypsin, indicating that these proteins use a common secretory pathway. Blood feeding triggers the secretion of vesicle contents into the midgut lumen, after which Ag-Aper1 is incorporated into the PM. Newly synthesized Ag-Aper1 protein was again detected within the midgut epithelial cells at 60 h after blood ingestion.

Induction of mosquitocidal activity in mice immunized with Anopheles gambiae midgut cDNA.

Vaccines that induce mosquito-killing (mosquitocidal) activity could substantially reduce the transmission of certain mosquito-borne diseases, especially vaccines against African malaria vectors, such as the mosquito Anopheles gambiae. To generate and characterize antimosquito immunity we immunized groups of mice with two individual A. gambiae midgut cDNAs, Ag-Aper1 (a secreted peritrophic matrix protein) and AgMuc1 (a midgut-bound mucin), and an A. gambiae midgut cDNA library from blood-fed mosquitoes. We observed significantly increased mortality among mosquitoes that fed on either the AgMuc1- or the cDNA library-immunized mice compared to that of controls, but no differences were observed among those fed on Ag-Aper1-immunized mice. Analysis of the humoral and cellular immune responses from mice showed that the induced mosquitocidal effect was associated with immune profiles characterized by elevated tumor necrosis factor alpha and gamma interferon cytokine levels and very low antibody titers. Furthermore, an additional immunization of cDNA library-immunized mice with midgut protein shifted immunity toward a Th2-type immune response, characterized by elevated antibody titers and high interleukin-5 and interleukin-10 cytokine levels; importantly, mosquitoes feeding on these mice exhibited no undue mortality. Finally, when immune sera was ingested by mosquitoes through a membrane feeder, no effect on mosquito mortality was observed, indicating that serum factors alone were not responsible for the mosquitocidal effect. Our results demonstrate that mosquitocidal immunity in mice can be consistently generated by midgut cDNA immunization and suggest this cDNA-induced mosquitocidal immunity is cell mediated.

The peritrophic matrix of hematophagous insects.

The peritrophic matrix (PM) is an extracellular envelope that lines the digestive tract of most insects. It is thought to play key roles in protecting insects from pathogens and facilitating digestion. Until recently, little information was available on the molecular composition of the PM. This review summarizes recent progress in the study of the PM from hematophagous insects, with emphasis on molecular and physiological aspects. Topics discussed include the presence of chitin and protein diversity in the PM, cloning and characterization of genes encoding PM proteins, PM permeability, and the role of the PM as a barrier for pathogens.

Characterization of the Hox gene cluster in the malaria vector mosquito, Anopheles gambiae.

The Hox genes play a central role in regulating development and are involved in the specification of cell fates along the anteroposterior axis. In insects and vertebrates, these genes are clustered and organized in an arrangement that is largely conserved across evolutionary lineages. By exploiting the sequence conservation of the homeobox, orthologues of the Hox genes Sex combs reduced (Scr), fushi tarazu (ftz), Antennapedia (Antp), Ultrabithorax (Ubx), and abdominal-A (abd-A) have been isolated from the malaria vector mosquito, Anopheles gambiae. These genes were first identified in Drosophila, where they achieve a high level of functional complexity, in part, by the use of alternative promoters, polyadenylation sites, and splicing to generate different protein isoforms. Preliminary analyses of the Anopheles Hox genes suggest that they do not achieve their functional complexity in the same manner. Using a combination of in situ hybridization to polytene chromosomes and chromosome walking, the Anopheles Hox genes have been localized to a single cluster in the region 19D-E on chromosome 2R, a situation distinct from that of Drosophila where the Hox complex is split into two clusters. This study, therefore, provides a framework for future comparative analyses of the structure, organization, and expression of developmental regulatory genes between the lower and higher Diptera. Moreover, the genes that have been isolated enhance the genetic and physical maps of chromosome 2R in this medically important mosquito species.

Relationships between blood pressure, oral contraceptive use and metabolic risk markers for cardiovascular disease.

Data from a previous study, designed to compare metabolic risk markers for cardiovascular disease in non-users and oral contraceptive (OC) users, were analysed to evaluate the influence of OC composition on blood pressure. Healthy, female volunteers (1189 women) either not using OC (non-users) or currently using one of six different combined formulations (users) were compared. Combinations studied contained 30-40 micrograms ethinyl estradiol combined with the progestins levonorgestrel, norethindrone (at two and three different doses, respectively) or desogestrel. After statistical standardisation to account for the significantly greater age of the non-users and longer duration of OC use amongst the levonorgestrel combination users, mean blood pressure was higher, compared with non-users, in users of monophasic or triphasic levonorgestrel combinations (systolic: +4.3 mmHg (p < 0.001) and +2.7 mmHg (p < 0.001), respectively; diastolic: +2.6 mmHg (p < 0.001) and +2.3 mmHg (p < 0.05), respectively). Blood pressures in users of monophasic norethindrone and desogestrel combinations were not significantly raised and there was no increase in the proportion of women with abnormal values. Diastolic and systolic blood pressures were positively associated with oral glucose tolerance test insulin response (r = 0.11 (p < 0.01) and r = 0.15 (p < 0.001), respectively) in users but not in non-users. Currently used OC containing norethindrone or desogestrel progestins have little impact on blood pressure. Their correlated reduction in impact on insulin concentrations, though small, suggests common mechanisms through which OC affect blood pressure and insulin.

PIP: The influence of oral contraceptive (OC) composition on blood pressure was investigated in 1189 healthy volunteers recruited from centers in London and southeast England. The mean age of the non-users was 32.5 years compared with 28.0 years among OC users. The OC users were currently taking one of six types of combined OCs containing 30-40 mcg of ethinyl estradiol combined with the progestins levonorgestrel (150 mcg or a 50-125 mcg triphasic), norethindrone (500 mcg, 1000 mcg, or a 500-1000 mcg triphasic), and desogestrel (150 mcg). After adjustment for age and duration of OC use, mean blood pressure was significantly higher compared to non-users in users of monophasic or triphasic levonorgestrel combinations (systolic, +4.3 and +2.7 mm Hg, respectively; diastolic, +2.6 and +2.3 mm Hg, respectively). There was no significant increase in blood pressure levels in users of monophasic norethindrone and desogestrel combinations. Diastolic and systolic blood pressures were significantly positively associated with oral glucose tolerance test insulin responses (r = 0.11 and -0.15, respectively) in OC users but not in non-users. These findings suggest that currently used low-estrogen dose OCs containing norethindrone or desogestrel have little effect on blood pressure. They further indicate that the typical profile recorded in OC users--elevated blood pressure, increased triglycerides, decreased high density lipoprotein cholesterol, increased insulin concentrations, and reduced insulin sensitivity--mainly reflect the independent effects of the contraceptive steroids rather than a single coordinated disturbance.

Danazol induces resistance to both insulin and glucagon in young women.

1. Danazol elevates plasma insulin, plasma glucagon and serum low-density lipoprotein concentrations and reduces the serum high-density lipoprotein concentration. 2. Associations between these disturbances were studied in 17 women receiving danazol therapy for endometriosis. Eleven women underwent intravenous glucose tolerance tests with measurement of plasma glucose, insulin, C-peptide and glucagon concentrations and modelling analysis of intravenous glucose tolerance test concentration profiles. Six women underwent glucagon sensitivity tests. Serum concentrations of lipids and lipoproteins were measured in all cases. 3. Danazol reduced the fasting plasma glucose and insulin concentrations, but markedly raised the fasting plasma glucagon concentration. The insulin and C-peptide responses to the intravenous glucose tolerance test were increased twofold and the net decrement in glucagon concentration was increased tenfold. The glucose response to the intravenous glucose tolerance test was unaffected. Insulin sensitivity was reduced by 55%. Both first-phase plasma insulin responsiveness and net first-phase pancreatic insulin secretion were increased; insulin half-life was prolonged. The glucose response to the glucagon sensitivity test was reduced on treatment. The calculated low-density lipoprotein cholesterol level rose by 20%, whereas high-density lipoprotein cholesterol level fell by 47%. None of these changes in serum lipoprotein levels correlated with changes in insulin metabolism. In general, metabolic changes normalized after 3 months. 4. Danazol increases the sensitivity of pancreatic insulin and glucagon secretion to glucose. Danazol-induced insulin and glucagon resistance could be due to receptor down-regulation resulting from hypersecretion of insulin and glucagon.

Lipid and carbohydrate metabolism in premenopausal women given subdermal estradiol implants.

Fifteen premenopausal women were studied before and 6 weeks after receiving subcutaneous implants of 100 mg estradiol. Serum estradiol levels doubled; increases were also seen in fasting serum total cholesterol and in high-density lipoprotein cholesterol (HDL). This increase was confined to the HDL2 subfraction, and was not reflected in the HDL apolipoproteins. Low density lipoprotein (LDL) cholesterol levels were unchanged, as were those of apolipoprotein B, the major protein component of LDL. Carbohydrate metabolism was assessed in a subgroup of 12 women. Estrogen implantation reduced fasting plasma glucose levels but did not alter the plasma glucose response to an oral glucose tolerance test. Plasma insulin levels were unchanged both in the fasted state and during the glucose tolerance test. Our findings indicate that parenteral administration of estradiol can alter lipid and carbohydrate metabolism in premenopausal women.

The effects of different formulations of oral contraceptive agents on lipid and carbohydrate metabolism.

BACKGROUND: Oral contraceptives can induce changes in lipid and carbohydrate metabolism similar to those associated with an increased risk of coronary heart disease, including increased serum triglyceride, low-density lipoprotein (LDL) cholesterol, and insulin levels and decreased high-density lipoprotein (HDL) cholesterol levels. In this study, we examined whether modification of the type or dose of progestin in oral-contraceptive preparations diminishes these changes. METHODS: We measured plasma lipoprotein levels and performed oral glucose-tolerance tests in a cross section of 1060 women who took one of nine types of oral contraceptives for at least three months and 418 women who took none. Seven of the contraceptive formulations contained various doses and types of progestin: levonorgestrel in low (150 micrograms), high (250 micrograms), and triphasic (50 to 125 micrograms) doses; norethindrone in low (500 micrograms), high (1000 micrograms), and triphasic (500 to 1000 micrograms) doses; and a new progestin, desogestrel, in one dose (150 micrograms). All seven contained 30 to 40 micrograms of ethinyl estradiol. Two additional formulations contained progestin alone. RESULTS: As compared with controls, women taking combination drugs did not have increased serum total cholesterol levels but did have increases of 13 to 75 percent in fasting triglyceride levels. Levels of LDL cholesterol were reduced by 14 percent in women taking the combination containing desogestrel and by 12 percent in those taking low-dose norethindrone. Levels of HDL cholesterol were lowered by 5 percent and 16 percent by the combinations containing low-dose and high-dose levonorgestrel, respectively; these decreases were due to reductions of 29 percent and 43 percent, respectively, in the levels of HDL subclass 2. The combination pill containing high-dose norethindrone did not affect HDL cholesterol levels, whereas that containing low-dose norethindrone increased HDL cholesterol levels by 10 percent. The desogestrel combination increased HDL cholesterol levels by 12 percent. Levels of apolipoproteins A-I, A-II, and B were generally increased by combination drugs. Depending on the dose and type of progestin, combination drugs were associated with plasma glucose levels on the glucose-tolerance test that were 43 to 61 percent higher than in controls, insulin responses 12 to 40 percent higher, and C-peptide responses 18 to 45 percent higher. Progestin-only formulations had only minor metabolic effects. CONCLUSIONS: The appropriate dose and type of progestin may reduce the adverse effects of oral contraceptives on many metabolic markers of risk for coronary heart disease. Progestin-only formulations or combinations containing desogestrel or low-dose norethindrone were associated wtih the most favorable profiles.

Metabolic effects of low-dose fluconazole in healthy female users and non-users of oral contraceptives.

1. Azole antifungal agents such as ketoconazole act by inhibiting cytochrome P-450 mediated sterol synthesis in the fungal cell membrane and thus have the potential to interfere with mammalian steroidogenesis. Fluconazole is a novel orally-effective antifungal triazole which has been reported to have more specific effects on the cytochrome P-450 enzymes involved in fungal sterol synthesis. 2. Due to the potential value of systemic antifungal agents in the treatment of infections commonly occurring in women, we assessed the effect of oral fluconazole on the metabolic profile of 18 healthy premenopausal women, 10 of whom were taking combined oral contraceptives (OC). Each woman acted as her own control, being studied both before and 21-28 days after fluconazole therapy (50 mg daily), in the luteal phase of consecutive menstrual cycles. 3. The endocrinological profile included measurement of serum oestradiol, progesterone, testosterone and sex hormone binding globulin (SHBG) concentrations, short tetracosactrin adrenal stimulation test and thyroid function tests. Carbohydrate metabolism was investigated by means of an oral glucose tolerance test with measurement of plasma glucose, insulin and C-peptide concentrations. Serum lipids, lipoproteins and apolipoproteins were analysed on samples taken after an overnight fast. 4. Minor biochemical changes associated with fluconazole treatment included increases in serum thyroxine and testosterone concentrations (but not in women taking OC as well as fluconazole) and in insulin and apolipoprotein B levels (but only in women taking OC as well as fluconazole). In general, these changes were small and of no clinical significance with the values remaining within the laboratory normal range. There were no adverse side-effects.(ABSTRACT TRUNCATED AT 250 WORDS)

PIP: The metabolic effects of the anti-fungal drug fluconazole were investigated in 18 women, 10 of whom were taking oral contraceptives, to examine whether this steroid antagonist has any effects primarily on hormone systems. The women, aged 29-40, took 50 mg fluconazole orally from Day 1 of their menstrual cycle for 21-28 days. Subjects kept a symptom diary, were tested weekly for hematological and liver function, and were checked for compliance by analyzing blood for drug by GLC. 5 women reported side effects: somnolence, dizziness, fatigue, increased appetite, headache (1) and nausea (1). No effects on liver function or menses were noted. The only significant findings were increases in serum thyroxine and testosterone in fluconazole-only subjects, and increases in insulin and apo-lipoprotein B in fluconazole-oral contraceptive subjects. Pills containing levonorgestrel were used by 9 women, desogestrel by 1. No significant differences were seen in estradiol, progesterone, sex-hormone-binding globulin, thyroid function, cortisol, glucose, C-peptide, cholesterol, triglycerides, lipoproteins. Thus it is unlikely that the short-term use of fluconazole for treatment of superficial mycoses, such as vulvovaginal candidiasis, will adversely affect steroid metabolism in women.

Determinants of bone density in normal women: risk factors for future osteoporosis?

Postmenopausal osteoporosis is an important public health problem in developed countries. Preventive treatment might effect a large reduction in the incidence, but this needs to be applied selectively to those women at increased risk. Loss of bone density results in an increased risk of fractures in the classical sites of vertebrae and proximal femur. A cross sectional study of bone density measurements was carried out in these sites in British women with a modern, precise densitometric technique. Possible predictors and risk factors for bone density were assessed in these women. Bone density was measured by dual photon absorptiometry in 284 apparently healthy women volunteers aged 21 to 68. The values obtained were similar to those obtained from equivalent studies performed in women in the United States. Peak adult bone density had been attained soon after the end of linear skeletal growth. Thereafter there was some decline with age in the proximal femur, but the major fall in bone density in all sites was related to the menopause. Other factors decreasing bone density, and hence increasing risk for osteoporosis, such as low body weight, alcohol and cigarette consumption, nulliparity, lack of previous use of oral contraceptives, and lack of regular exercise, seemed to be important. None, however, could predict satisfactorily women at future risk for osteoporosis. Direct measurements of bone density in the clinically relevant sites are necessary to determine which women should received preventive treatment for postmenopausal osteoporosis. This would help make such treatment more cost effective.