Molecular characterizations of three distinct Babesia gibsoni rhoptry-associated protein-1s (RAP-1s).

Three cDNAs encoding rhoptry-associated protein 1 (RAP-1) homologues were found in the Babesia gibsoni EST database. Based on similarities to BgRAP-1a, which was identified previously by serological screening of a cDNA merozoite library, the two new genes were designated BgRAP-1b (33.7%) and BgRAP-1c (57%). Mice antiserum raised against each recombinant protein reacted specifically with B. gibsoni parasites as determined by Western blotting, which showed native molecular sizes of the BgRAP-1a (51 kDa), BgRAP-1b (53 kDa) and BgRAP-1c (47 kDa) consistent with predictable molecular weights. Immunofluoresence using these antibodies revealed localization of all BgRAP-1s within the matrix of merozoites; however, BgRAP-1a appeared to diverge from the other two when it was found secreted into the cytoplasm of infected erythrocytes. Apical localization of all 3 BgRAP-1s during the extracellular stage of the parasite combined with their ability to bind a canine erythrocyte membrane fraction was suggestive of a role for these proteins in erythrocyte attachment. Lastly, the ability of these recombinant proteins to be used as diagnostic reagents was tested by ELISA and the sensitivities of BgRAP-1a and BgRAP-1c were found increased through N-terminal truncation. Taken together, our data suggest divergent roles for the 3 BgRAP-1s in the merozoite stage of B. gibsoni.

Molecular and immunological characterization of Babesia gibsoni and Babesia microti heat shock protein-70.

Serological immunoscreening was used to identify a gene encoding heat shock protein-70 from Babesia gibsoni (BgHSP-70) that showed high homology with HSP-70s from other apicomplexan parasites. This gene corresponded to a full-length cDNA containing an open reading frame of 1968 bp predicted to result in a 70-kDa mature protein consisting of 656 amino acids. Analysis of the expression levels of BgHSP-70 indicated elevated transcription from cultured parasites incubated at 40 degrees C for 1 h, but not at 30 degrees C. Interestingly, antiserum raised against recombinant BgHSP-70 protein reacted specifically not only with a 70-kDa protein of B. gibsoni but also with a corresponding native protein of B. microti (BmHSP-70), indicating the high degree of conservation of this protein. The BmHSP-70 gene was then isolated and characterized and the immunoprotective properties of recombinant BgHSP-70 (rBgHSP-70) and rBmHSP-70 were compared in vitro and in vivo. Both proteins had potent mitogenic effects on murine and canine mononuclear cells as evidenced by high proliferative responses and IFN-gamma production after stimulation. Immunization regimes in BALB/c and C57BL/6 mice using rBgHSP-70 and rBmHSP-70 elicited high antibody levels, with concurrent significant reductions in peripheral parasitaemias. Taken together, these results emphasize the potential of HSP-70s as a molecular adjuvant vaccine.

Ethanol stimulates glycogenolysis and inhibits both glycogenesis via gluconeogenesis and from exogenous glucose in perfused rat liver.

AIMS: Although it is commonly recognized that ethanol suppresses gluconeogenesis, the influence of alcohol intake on blood glucose levels remains controversial. Ethanol may act on both glucose production and glucose consumption in the liver. Thus, we studied each effect of ethanol on glucose oxidation, gluconeogenesis, glycogenesis and glycogenolysis in the liver. METHODS: The rat liver was isolated and cyclically perfused with a medium containing 50 mmol/l ethanol. RESULTS: Ethanol enhanced 14C-glucose oxidation in the liver from 1.09 +/- 0.11 to 1.41 +/- 0.14 micromol for 20 min (p < 0.05). Gluconeogenesis from 14C-lactate was markedly reduced by ethanol from 8.0 +/- 1.3 to 1.5 +/- 0.6 micromol for 12 min (p < 0.01). Ethanol increased glycogenolysis (net hepatic glucose output, 0.47 +/- 0.10 vs. 0.22 +/- 0.04 mmol/30 min, p < 0.01), and then decreased hepatic glycogen content (179 +/- 38 vs. 273 +/- 39 mg in the presence of 1 mU/ml insulin after 30 min of perfusion, p < 0.05). Ethanol decreased the direct glycogenesis from 14C-glucose from 0.55 +/- 0.08 to 0.33 +/- 0.05 micromol per 100 mg glycogen for 30 min (p < 0.01). Ethanol inhibited the indirect glycogenesis from 14C-lactate from 0.21 +/- 0.04 to 0.09 +/- 0.01 micromol per 100 mg glycogen for 30 min (p < 0.01). DISCUSSION: The influence of ethanol on the blood glucose regulation by the liver seems to be different between fasted and fed states. Namely, ethanol has both the hypoglycemic effects through decreased gluconeogenesis and increased glucose oxidation and the hyperglycemic effects through decreased glycogenesis and increased glycogenolysis.

Gliclazide at a lower concentration than therapeutic dose increases the sensitivity of insulin secretion to glucose in perfused rat pancreas.

OBJECTIVES: We studied the difference between effects of therapeutic dose and sub-therapeutic dose of gliclazide on the glucose-induced insulin secretion. METHODS: The normal rat pancreas was isolated and perfused with Krebs-Ringer buffer containing 1-14 mmol/l glucose. Influcences of 0.25 and 2.5 microg/ml gliclazide on the glucose concentration-insulin secretion curve was examined. RESULTS: Gliclazide at 0.25 microg/ml significantly potentiated 5-8 mmol/l glucose-induced insulin secretion (2.5 +/- 0.5 vs 1.0 +/- 0.3 mU for 15 min at 6.5 mmol/l glucose, P<0.01), but did not give influence on either 1-3 or 10-14 mmol/l glucose-induced insulin secretion. The glucose concentration, at which half-maximal insulin secretion was observed, was lower with gliclazide (5.9 mmol/l) than in the control (7.5 mmol/l). Gliclazide at 2.5 microg/ml markedly increased the maximally glucose-stimulated insulin secretion from 3.9 +/- 0.5 mU for 15 min in the control to 6.6 +/- 0.7 mU for 15 min (P<0.01). The half-maximal insulin secretion was observed at a lower glucose concentration (5.0 mmol/l) than in the absence of gliclazide. CONCLUSION: Gliclazide in sub-therapeutically low dose has different effects on insulin secretion from in therapeutic dose, namely sharpens the insulin secretion sensitivity to glucose with no influence on the maximal insulin secretion. It is possible that low doses of gliclazide might be of interest in some type 2 diabetics whose main pathophysiology is the blunting of insulin secretion response to hyperglycemia.

Glucagon is paradoxically secreted at high concentrations of glucose in rat pancreas perfused with diazoxide.

To study the role of B-cells in the regulation of glucagon secretion by glucose, the rat pancreas was perfused with 0.4 mmol/l diazoxide. Perfusate glucose was 5 mmol/l of a basal concentration, and then was decreased to 1 mmol/l, or was increased to 15 mmol/l. Insulin secretion was suppressed by diazoxide below the detectable level at each glucose concentration. Glucagon secretion was increased two-fold during the glucopenic perfusion without diazoxide, but was not changed at a low glucose concentration in the presence of diazoxide. During the glucose-excessive perfusion for 15 min, glucagon secretion was lowered from 0.69 +/- 0.17 pmol at 5 mmol/l glucose to 0.36 +/- 0.10 pmol at 15 mmol/l glucose (p < 0.05) without diazoxide, whereas that was inversely increased from 0.55 +/- 0.14 at 5 mmol/l glucose to 0.85 +/- 0.13 pmol at 15 mmol/l glucose (p < 0.05) in the presence of diazoxide. These results suggest that appropriate insulin secretion is necessary for the normal responses of glucagon secretion to hypoglycemia and hyperglycemia in the non-diabetic rat pancreas.

Differences in the effects of 20 K- and 22 K-hGH on water retention in rats.

Antidiuretic actions induced by two growth hormone (GH) isoforms (20 K- and 22 K-hGH; 0.2 and 2.0 mg/kg) were evaluated in rats, as fluid retention may cause oedema, one of the adverse effects of GH. Both GH isoforms (2.0 mg/kg) suppressed urine excretion in hypophysectomized rats (P< 0.01), but only the 22 K-hGH isoform (2.0 mg/kg) suppressed urine excretion in intact rats (P< 0.01). In addition, prolactin (PRL) suppressed urine excretion in intact rats (P< 0.05). In conclusion, 20 K-hGH has less potency in causing urine retention than 22 K-hGH and since 20 K-hGH is missing 15 amino acids found in 22 K-hGH, these amino acids may be important for the antidiuretic action of GH. Since prolactin suppressed urine excretion, a part of the antidiuretic action of GH may be related to PRL-R activation.

Advanced glycation end products-driven angiogenesis in vitro. Induction of the growth and tube formation of human microvascular endothelial cells through autocrine vascular endothelial growth factor.

This study was undertaken to determine whether and how advanced glycation end products (AGE), senescent macroproteins accumulated in various tissues under hyperglycemic states, cause angiogenesis, the principal vascular derangement in diabetic microangiopathy. We first prepared AGE-bovine serum albumin (BSA) and anti-AGE antiserum using AGE-RNase A. Then AGE-BSA was administered to human skin microvascular endothelial cells in culture, and their growth was examined. The AGE-BSA, but not nonglycated BSA, was found to induce a statistically significant increase in the number of viable endothelial cells as well as their synthesis of DNA. The increase in DNA synthesis by AGE-BSA was abolished by anti-AGE antibodies. AGE-BSA also stimulated the tube formation of endothelial cells on Matrigel. We obtained the following evidence that it is vascular endothelial growth factor (VEGF) that mainly mediates the angiogenic activities of AGE. (1) Quantitative reverse transcription-polymerase chain reaction analysis of poly(A)+ RNA from microvascular endothelial cells revealed that AGE-BSA up-regulated the levels of mRNAs for the secretory forms of VEGF in time- and dose-dependent manners, while endothelial cell expression of the genes encoding the two VEGF receptors, kinase insert domain-containing receptor and fms-like tyrosine kinase 1, remained unchanged by the AGE treatment. Immunoprecipitation analysis revealed that AGE-BSA did increase de novo synthesis of VEGF. (2) Monoclonal antibody against human VEGF completely neutralized both the AGE-induced DNA synthesis and tube formation of the endothelial cells. The results suggest that AGE can elicit angiogenesis through the induction of autocrine vascular VEGF, thereby playing an active part in the development and progression of diabetic microangiopathies.

Age-related changes in the numbers of mammotrophs, somatotrophs and mammosomatotrophs in the anterior pituitary gland of female rats: a flow cytometric study.

Age-related changes in the numbers of mammotrophs, somatotrophs, mammosomatotrophs, and the cells of other types in the anterior pituitary of female Wistar rat were measured by flow cytometry. The mammotrophs increased with age, and the somatotrophs decreased with senescence. The mammosomatotrophs increased remarkably in senescent rats, and these cells of the rats older than 21 months were about 10 times more than those of 3, and 12-13 months old rats. This result indicates that the stability of gene expression in cell differentiation is reduced in the aging process of the anterior pituitary.

Effect of angiotensin II on the proliferation of mammotrophs from the adult rat anterior pituitary in culture.

We measured the influence of prolactin-releasing neuropeptides on mammotroph proliferation in cultures of rat adenohypophysis cells using flow cytometry. Angiotensin II (AII) increased mammotroph proliferation. Other peptides with hormone-releasing activities did not promote growth. Tamoxifen inhibited mammotroph proliferation in control and AII-containing cultures and the inhibition was reversed with beta-estradiol. Saralasin, an AII receptor antagonist, suppressed not only AII-induced mammotroph proliferation but also luteinizing hormone-releasing hormone (LHRH)-induced proliferation. These results suggest that hypothalamic LHRH stimulates AII release from gonadotrophs and that AII, with estrogen, controls mammotroph proliferation in rat pituitary.

Immunocytochemical demonstration of prolactin interaction with choroid plexus in aging and acute hyperprolactinemia.

Prolactin (PRL) exerts a direct effect on the central nervous system, reaching the PRL-responsive brain regions via cerebro-spinal fluid (CSF). The hormone enters the CSF by a specific receptor-mediated transport mechanism that is localized on the epithelium of the choroid plexus (CP) of brain ventricles. PRL interactions with the CP in aging were examined in young (3-month) and old (27-month) female Wistar rats using immunocytochemistry (immunogold technique). The enhancement of PRL uptake by the CP in animals at both ages was achieved by the modelling of acute hyperprolactinemia. A great age-related difference was found in the intensity of immunocytochemical reaction under activated conditions, the uptake of PRL by CP being significantly higher in young animals than in old. The character of the colloidal gold particle distribution in different components of CP epithelial cells appeared to be the same in both age groups. The weakening of PRL-transporting capacity in the CP of old animals may constitute one aspect of the alteration of neuroendocrine regulation in the CP-CSF system that occurs during aging.

[A müllerian duct cyst treated by injection of minocycline: a case report].

A 29-year-old male was admitted for the examination of left flank pain. Abdominal ultrasonography revealed a cystic mass beneath the urinary bladder. Computed tomographic scan, magnetic resonance imaging and transrectal ultrasonography showed that this cystic mass was located between the bladder and the prostate. DIP, urethrocystography and seminal vesiculography demonstrated no communication between this mass and the urinary and the seminal tract. From these findings it was diagnosed a müllerian duct cyst. We obtained 15 ml of clear yellowish fluid which contained no sperm by fine needle aspiration under ultrasound guidance and injected minocycline for sclerotherapy. Left flank pain had disappeared and repeated ultrasonography showed a small cystic lesion. However, the size of the cyst was unchanged for six months after discharge. Twenty-five müllerian duct cysts reported in the Japanese literature were reviewed.

Age-related changes in prolactin secretion and the population of mammotrophs in the rat: a longitudinal study.

The concentration of prolactin (PRL) in the blood increases with aging in the rat, and pituitary prolactinomas often develop in aged female rats. We measured blood PRL levels in the course of development of hyperprolactinemia and prolactinomas in individual rats by serial sampling of the blood of living rats. The pituitaries were examined upon the death of the rats to detect prolactinomas by immunocytochemical staining. Hyperprolactinemia in rats without pituitary tumors was inevitably followed by the development of a prolactinoma within 3 months. Rats which had no tumors at the time of death had low PRL levels throughout their lifespan. Counting of anterior pituitary cells after immunocytochemical staining for electron microscopy indicated that the number of mammotrophs increased and that the number of somatotrophs decreased in rats with hyperprolactinemia. Based on these results, we conclude that age-related hyperprolactinemia in female rats represents a precancerous state for prolactinoma.

Growth hormone-releasing factor (GRF) suppresses the in vitro proliferation of mammotrophs from the adult rat.

In order to examine the hypothalamic control of acidophilic proliferation, anterior pituitary cells from adult female rats were cultured with or without rat growth hormone-releasing factor fragment 1-29 (GRF-29). Changes in the numbers of mammotrophs and somatotrophs during culture were measured by immunocytochemical staining. The addition of GRF suppressed the increase in the number of mammotrophs even at the very low concentration of 10(-12) M. The number of somatotrophs increased in the medium containing GRF. The increase in mammotroph number was blocked by cytosine arabinoside, a mitotic inhibitor. GRF had no effect on the in vitro proliferation of fibroblasts. These results indicate the important role of hypothalamic GRF in the differential growth and secretion of the acidophils in vivo.

Changes in basal secretion rates of thyroxine and 3,3',5-triiodothyronine from the thyroid gland during aging of the rat.

The present experiments were carried out to examine age-related changes in the basal secretion rates of both thyroxine (T4) and 3,3',5-triiodothyronine (T3) from the thyroid gland. The experiments were performed on male Wistar rats of three different ages, i.e., (1) adult rats of 6-8 months old, (2) middle-aged rats of 25-26 months old, and (3) aged rats of 28-30 months old. The rats were anesthetized with 1.0% halothane. The thyroid venous blood as well as systemic arterial blood was collected and secretion rates of both immunoreactive T4 (iT4) and T3 (iT3) from the thyroid gland were calculated from the differences in concentrations of iT4 and iT3 in these two blood plasmas, and from the flow rate of thyroid venous blood plasma. The secretion rates of thyroid iT4 in the three different age groups were as follows: 483 +/- 68 pg/min (mean +/- S.E.) in the adult rats, 598 +/- 104 pg/min in the middle-aged rats, and 491 +/- 150 pg/min in the aged rats. There were no significant differences among the secretion rates of thyroid iT4 in these rats. The secretion rates of thyroid iT3 in the groups were as follows: 36.2 +/- 7.5 pg/min in the adult rats, 58.9 +/- 13.9 pg/min in the middle-aged rats, and 61.3 +/- 7.6 pg/min in the aged rats. The secretion rates of thyroid iT3 in both middle-aged and aged rats were approximately 1.6-1.7 times as high as the value in adult rats (p less than 0.05). These results indicate that the thyroid secretion of iT4 is well maintained, while that of iT3 increases during aging.

A new human male diploid cell strain, TIG-7: its age-related changes and comparison with a matched female TIG-1 cell strain.

A new human diploid cell strain, TIG-7, which has the male karyotype, was established and characterized. Isozyme and histocompatibility typing of the cell strain was performed. The average in vitro life span of the cells is 73 population doublings. Changes in cell volume, doubling time, saturation density, the efficiency of cell attachment, plating efficiency, and relative DNA content were examined during in vitro cellular aging. Hydrocortisone slightly prolongs the life span of the cell strain when the hormone is administered to the cultures during middle passages. The age-related changes in the parameters of TIG-7 are not appreciably different from those of the previously established TIG-1 cell strain. These results show that this cell strain is useful for research on cellular aging; further profit is anticipated from research using a combination of these two sexually different cell strains.

Appearance of the terminal senescent cell population in human diploid fibroblasts analyzed by flow cytometry.

We studied changes in the distribution pattern of relative RNA content during the in vitro aging of TIG-3 cells by flow cytometry (FACS III). Propidium iodide (PI) does not stain total cellular RNA, but it intercalates specifically into double-helical regions of both DNA and RNA. In applying this principle to RNA, we stained double-stranded RNA (dsRNA) in whole cells with PI after DNA digestion with DNase. The results showed that dsRNA distribution patterns were relatively constant at 7-75 population doublings (PD) but were significantly altered after 77 PD. The distribution patterns were similar as those for cell volume measured with a Coulter Counter. The total cellular dsRNA contents increased linearly at the senescent phase of their in vitro life span. In contrast, the mean dsRNA contents (50% dsRNA contents) rapidly increased to 77-79 PD, but decreased somewhat at 81-83 PD. Two-dimensional histograms of the dsRNA contents versus cell size were little altered from 25 PD to 75 PD. However, a population with relatively larger cell volume and weaker fluorescence intensity appeared and increased after 79 PD. This cell population group may be categorized as "terminal senescent cells" that no more divide in respect that the dsRNA content decreases in spite of the increase of total RNA content.

Histology and survival in age-delayed low-tryptophan-fed rats.

Diets containing tryptophan in concentrations 30 and 40 percent of those fed to controls from weaning to 24-30 months or more, can delay aging in Long-Evans female rats. Mortality among low-tryptophan-fed rats was greater in the juvenile period, but substantially less than controls at late ages. Histological biomarkers of aging were also delayed after tryptophan restriction in some organs (liver, heart, uterus, ovary, adrenal and spleen) but not in others (kidney, lung, aorta). Brain serotonin levels were low in tryptophan-deficient rats but showed remarkable capacity for rehabilitation. Effects on early and late mortality and brain levels of serotonin were proportional to the severity of the restriction.

Changes in negative surface charge of human diploid fibroblasts, TIG-1, during in vitro aging.

The electrophoretic mobility of human diploid fibroblasts, TIG-1, was studied at different passages. The net negative surface charge of the cells decreased from -1.658 +/- 0.108 micron/s/V/cm at an early passage (15 population doublings, PD) to -1.173 +/- 0.116 at the final passage (67 PD) in 1/15 M phosphate buffer supplemented with 5.4% glucose. The decrease was slow at 15-45 PD, but was rapid at 45-67 PD. The net negative surface charge of small cells in the late passage populations was not different from that of larger cells in this population, and was significantly lower than that of small cells in the middle passage populations. The distribution of the mobilities of cells in each passage was independent of the size of the individual cells, and the mean value was distinct for the passage number. The viability of the cells was retained during the assay of electrophoretic mobility under these conditions. These results indicate that the net negative surface charge of human diploid fibroblasts represents a cell surface maker for in vitro cellular age in the population.

Effects of chronic hyperthyroidism on the lifespan of the rat.

The life durations of hypo- and hyperthyroid Wistar rats were measured under clean conventional conditions. The amount of exogenous T4 (thyroxine), which is sufficient to elevate T4 levels in the blood, decreased with age. The rats which were made hypothyroid by the neonatal T4 treatment had a longer lifespan than control. The lifespans of hyperthyroid rats, to which T4 solutions were given as drinking water during either the first or the second half of the life period, were shorter than control. The life-shortening effect of T4 was not detected when T4 was administered to already aged animals. These results indicate that the effect of T4 administration is not due to the direct promotion of the diseases which cause the death, but to the acceleration of aging during the young or middle-aged period.

Pituitary-thyroid activity and longevity in neonatally thyroxine-treated rats.

Wistar rats were made hypothyroidic by intraperitoneal thyroxine (T4) injection during the first 10 days of neonatal life. Levels of T4, 3,3',5-triiodothyronine (T3), thyroid-stimulating hormone (TSH) and prolactin in the blood of these rats were measured by radioimmunoassay. The T4 levels are about two-thirds of control values up to 20 months of age. T3 level is low only at a young age. TSH level shows no significant difference from control, but is about half that of control after the stimulation of secretion by 6-propyl-2-thiouracil. The level of prolactin is much higher in the T4-treated group than in controls. In male rats, life duration of hypothyroid rats was longer than control by about 4 months. The life extension effect of hypothyroidism was observed also in females, although the difference was smaller than that in males. The concentration of T4 in the blood of male rats is higher than females, and the decrease in T4 level by neonatal T4 treatment is also more marked in males.