Mice lacking acylation stimulating protein (ASP) have delayed postprandial triglyceride clearance.

Acylation stimulating protein (ASP) is a 76 amino acid fragment of the third component of complement (C3) which is generated by the interaction of adipsin and factor B with C3. In vitro studies have shown that ASP can markedly increase triglyceride synthesis in adipocytes. To test the ASP pathway in vivo, C3-deficient mice, and therefore ASP-deficient mice, were generated and oral fat loads were conducted in wild-type (C3+/+) and mutant (C3-/-) animals. The principal results were: 1) postprandial triglyceride clearance was significantly delayed in mutant compared to wild-type mice; 2) this difference was more pronounced in males compared to females; 3) in both males and females, the differences were more pronounced in the second half of the postprandial period; 4) fasting and postprandial free fatty acid (FFA) were higher in C3(-/-) than in C3(+/+) males; and 5) intraperitoneal administration of ASP accelerated triglyceride clearance in C3(-/-) males. The data are consistent therefore, with the hypothesis that the ASP pathway is an important physiologic determinant of normal postprandial triglyceride clearance.     

Mice lacking the UBC4-testis gene have a delay in postnatal testis development but normal spermatogenesis and fertility

Activation of ubiquitination occurs during spermatogenesis and is dependent on the induction of isoforms of the UBC4 family of ubiquitin-conjugating enzymes. The UBC4-testis isoform is testis specific, is induced in round spermatids, and demonstrates biochemical functions distinct from a ubiquitously expressed isoform UBC4-1. To explore further the function of UBC4-testis, mice bearing inactivation of this gene were produced. Homozygous (-/-) mice showed normal body growth and fertility. Although testis weight and morphology were normal in testes from adult mice, examination of young mice during the first wave of spermatogenesis revealed that testes were approximately 10% smaller in weight at 40 and 45 days of age but had become normal at 65 days of age. Overall protein content, levels of ubiquitinated proteins, and ubiquitin-conjugating activity did not differ between wild-type and homozygous (-/-) mice. Spermatid number, as well as the motility of spermatozoa isolated from the epididymis, was also normal in homozygous (-/-) mice. To determine whether the germ cells lacking UBC4-testis might be more sensitive to stress, testes from wild-type and knockout mice were exposed to heat stress by implantation in the abdominal cavity. Testes from both strains of mice showed similar rates of degeneration in response to heat. The lack of an obvious phenotype did not appear to be due to induction of other UBC4 isoforms, as shown by two-dimensional gel immunoblotting. Our data indicate that UBC4-testis plays a role in early maturation of the testis and suggest that the many UBC4 isoforms have mixed redundant and specific functions.     

Thiamin dynamics during the adult life cycle of Atlantic salmon (Salmo salar)

Thiamin is an essential water-soluble B vitamin known for its wide range of metabolic functions and antioxidant properties. Over the past decades, reproductive failures induced by thiamin deficiency have been observed in several salmonid species worldwide, but it is unclear why this micronutrient deficiency arises. Few studies have compared thiamin concentrations in systems of salmonid populations with or without documented thiamin deficiency. Moreover, it is not well known whether and how thiamin concentration changes during the marine feeding phase and the spawning migration. Therefore, samples of Atlantic salmon (Salmo salar) were collected when actively feeding in the open Baltic Sea, after the sea migration to natal rivers, after river migration, and during the spawning period. To compare populations of Baltic salmon with systems without documented thiamin deficiency, a population of landlocked salmon located in Lake Vänern (Sweden) was sampled as well as salmon from Norwegian rivers draining into the North Atlantic Ocean. Results showed the highest mean thiamin concentrations in Lake Vänern salmon, followed by North Atlantic, and the lowest in Baltic populations. Therefore, salmon in the Baltic Sea seem to be consistently more constrained by thiamin than those in other systems. Condition factor and body length had little to no effect on thiamin concentrations in all systems, suggesting that there is no relation between the body condition of salmon and thiamin deficiency. In our large spatiotemporal comparison of salmon populations, thiamin concentrations declined toward spawning in all studied systems, suggesting that the reduction in thiamin concentration arises as a natural consequence of starvation rather than to be related to thiamin deficiency in the system. These results suggest that factors affecting accumulation during the marine feeding phase are key for understanding the thiamin deficiency in salmonids.     

Increased apoptosis during neonatal brain development underlies the adult behavioral deficits seen in mice lacking a functional paternally expressed gene 3 (Peg3)

Inactivation of the maternally imprinted, paternally expressed gene 3 (Peg3) induces deficits in olfactory function, sexual and maternal behaviors, oxytocin neuron number, metabolic homeostasis and growth. Peg3 is expressed in a number of developing hypothalamic and basal forebrain structures and is a component of the P53 apoptosis pathway. Peg3 inactivation in neuronal cell culture lines inhibits P53 mediated apoptosis, which is important in the early postnatal development and sexual differentiation of the brain. In this study, we investigated the effect of inactivating the Peg3 gene on the incidence of caspase 3 positive cells (a marker of apoptosis) in 4‐ and 6‐day postpartum mouse brain. Inactivating the Peg3 gene resulted in an increase in the incidence of total forebrain caspase 3 positive cells at 4 and 6 days postpartum. Increases in specific neuroanatomical regions including the bed nucleus of the stria terminalis, nucleus accumbens, caudate putamen, medial pre‐optic area, arcuate nucleus, medial amygdala, anterior cortical and posteriodorsal amygdaloid nuclei, were also observed. In wild‐type mice, sex differences in the incidence of caspase 3 positive cells in the medial amygdala, bed nucleus of the stria terminalis, nucleus accumbens, arcuate nucleus and the M2 motor cortex, were also observed. This neural sex difference was ameliorated in the Peg‐3 mutant. These findings suggest that the neuronal and behavioral deficits seen in mice lacking a functional Peg3 gene are mediated by increases in the incidence of early neonatal apoptosis in neuroanatomical regions important for reproductive behavior, olfactory and pheromonal processing, thermoregulation and reward. © 2009 Wiley Periodicals, Inc. Develop Neurobiol, 2009     

Mice lacking the poly(ADP-ribose) polymerase gene are resistant to pancreatic beta-cell destruction and diabetes development induced by streptozocin

Human type 1 diabetes results from the selective destruction of insulin-producing pancreatic beta cells during islet inflammation. Cytokines and reactive radicals released during this process contribute to beta-cell death. Here we show that mice with a disrupted gene coding for poly (ADP-ribose) polymerase (PARP-/- mice) are completely resistant to the development of diabetes induced by the beta-cell toxin streptozocin. The mice remained normoglycemic and maintained normal levels of total pancreatic insulin content and normal islet ultrastructure. Cultivated PARP-/- islet cells resisted streptozocin-induced lysis and maintained intracellular NAD+ levels. Our results identify NAD+ depletion caused by PARP activation as the dominant metabolic event in islet-cell destruction, and provide information for the development of strategies to prevent the progression or manifestation of the disease in individuals at risk of developing type 1 diabetes.     

Mice lacking the Nkx6.2 (Gtx) homeodomain transcription factor develop and reproduce normally

The Nkx homeobox genes are expressed in a variety of developing tissues and have been implicated in controlling tissue patterning and cell differentiation. Expression of Nkx6.2 (Gtx) was previously observed in the embryonic neural tube, testis, and differentiating oligodendrocytes. To investigate the role of Nkx6.2 in the control of cell specification and differentiation, we generated mice with null mutations in Nkx6.2 using the standard gene targeting approach. Null mutant mice were viable and fertile without apparent histological and immunohistochemical changes in the central nervous systems and testis. The absence of detectable phenotypes suggests a redundant function of Nkx6.2 in mouse development.     

Melanocortin-4 receptor (MC4R) genotypes have no major effect on fatness in a Large White x Wild Boar intercross

The melanocortin-4 receptor (MC4R), a G-protein coupled receptor, is implicated in mediating the effect of leptin on food intake and energy balance. A previous candidate gene study reported an association between an MC4R missense mutation (Asp298Asn) and fatness, growth and feed intake in pigs. To assess this association further, we analysed the segregation of this missense mutation in relation to variation in fatness traits using a Wild Boar x Large White intercross. The Wild Boar and Large White founders were homozygous for different MC4R alleles. The MC4R was assigned to the expected region on pig chromosome 1. The statistical evaluation did not reveal any indication of a significant effect on fatness related traits in this pedigree.     

Benzalkonium chloride (BAK) induces apoptosis or necrosis, but has no major influence on the cell cycle of Jurkat cells

Benzalkonium chloride (BAK) is a cationic detergent with a very slow turnover. Because of its strong antibacterial activities, BAK is widely used especially in dentistry and ophthalmology. It is the most commonly used preservative in topical ophthalmic medications. Due to chronicity and widespread use of such treatments, BAK's side effects are of great importance. BAK toxicity for adherent cells, probably related to its pro-oxidative activities, is time- and dose-dependent. Although lymphocytes often infiltrate superficial eye tissues, the BAK influence on them is yet to be established. The aim of this study was to check BAK cytotoxicity on T lymphocytic Jurkat line cells and to verify the suggestion that BAK can induce G2M cell blocks. A dose- and time-dependent cytotoxic effect of BAK on lymphoid cells in relatively low concentrations was shown in this study. In lower concentrations, it shows a moderate apoptotic and minimal antiproliferative effect on Jurkat cells, while in higher concentrations it shows a rapid necrotic effect. No G2M cell blocks were observed. Our findings could suggest lymphoid dysfunction during intensive, prolonged topical BAK treatment, even at dosages relatively non-toxic to epithelial eye cells.     

Expression of the presenilin-like signal peptide peptidase (SPP) in mouse adult brain and during development

Recently, a new member of the presenilin family was identified as an aspartyl protease that cleaves signal peptides within hydrophobic domains, and was, therefore, named signal peptide peptidase (SPP). We isolated cDNAs coding for mouse and human orthologues of SPP. The human gene spans 55 kilobases on chromosome 20q11.21. The SPP-protein is encoded in mouse and man by 12 exons. The highly conserved intron/exon-structure in the SPP/presenilin family hints at a common precursor. Northern blot and in situ hybridization analysis revealed a widespread expression of SPP in many tissues. A distinct pattern of expression in the mature murine brain and during development indicates that SPP plays an important role in the establishment and maintenance of the nervous system. We prepared an antiserum against the carboxy-terminal domain of SPP, which is highly conserved between species. It reacted specifically, both in western blots and in immunocytochemical preparations, with SPP from various mammalian origins. The antiserum was used to demonstrate that SPP is oriented in the membrane of the endoplasmic reticulum with its carboxy-terminal tail extending into the cytosol.     

Mortality of Apatania fimbriata (Insecta: Trichoptera) during embryonic, larval and adult life stages

1. Egg masses, oviposition site preferences, and abiotic and biotic factors causing mortality during embryonic, larval and adult life stages of Apatania fimbriata were studied. Laboratory investigations provided information on the temperature dependence of embryonic development, measured as an increase in egg volume.
2. A. fimbriata laid hemispherical egg masses, consisting of a transparent matrix containing a mean of 208 eggs. Egg masses were laid on stones situated just above the water surface in dark cavities in the stream bank.
3. Two hundred egg masses were mapped and individually monitored during embryonic development. There was no significant correlation between mortality during embryonic development and any of the abiotic parameters measured. First-instar larvae of Osmylus fulvicephalus consumed developing eggs, and chironomids preyed on newly hatched larvae.
4. A mean of seventy-two females emerged per metre of stream. Mortality during the 1993/94 life cycle was measured as a percentage of the potential number of eggs laid. Female mortality between emergence and oviposition was ≈ 80%. Eight per cent of individuals were lost during embryonic development. Larval mortality to emergence in 1994 was 11.3%. This indicates that the terrestrial life stage is probably decisive in the regulation of A. fimbriata populations.
5. Duration of embryogenesis at constant temperatures (4–20 °C) in the laboratory was described best by a negative exponential function. This species is cold stenothermal and there was no hatching success at 20 °C.
6. Egg volumes during embryonic development increased sigmoidally over time.     

Mortality of Apatania fimbriata (Insecta: Trichoptera) during embryonic, larval and adult life stages

1. Egg masses, oviposition site preferences, and abiotic and biotic factors causing mortality during embryonic, larval and adult life stages of Apatania fimbriata were studied. Laboratory investigations provided information on the temperature dependence of embryonic development, measured as an increase in egg volume.
2. A. fimbriata laid hemispherical egg masses, consisting of a transparent matrix containing a mean of 208 eggs. Egg masses were laid on stones situated just above the water surface in dark cavities in the stream bank.
3. Two hundred egg masses were mapped and individually monitored during embryonic development. There was no significant correlation between mortality during embryonic development and any of the abiotic parameters measured. First-instar larvae of Osmylus fulvicephalus consumed developing eggs, and chironomids preyed on newly hatched larvae.
4. A mean of seventy-two females emerged per metre of stream. Mortality during the 1993/94 life cycle was measured as a percentage of the potential number of eggs laid. Female mortality between emergence and oviposition was ≈ 80%. Eight per cent of individuals were lost during embryonic development. Larval mortality to emergence in 1994 was 11.3%. This indicates that the terrestrial life stage is probably decisive in the regulation of A. fimbriata populations.
5. Duration of embryogenesis at constant temperatures (4–20 °C) in the laboratory was described best by a negative exponential function. This species is cold stenothermal and there was no hatching success at 20 °C.
6. Egg volumes during embryonic development increased sigmoidally over time.     

Immunocytochemical and electrophoretic studies on the localization of the major haemolymph proteins (ceratitins) inCeratitis capitata during development

Summary The developmental profile of the major haemolymph proteins (ceratitins) inCeratitis capitata was studied. Ceratitin concentration in the haemolymph decreases dramatically during the last days of pupal life, while the amounts of ceratitins in whole organism extracts remain unchanged. By electrophoretic, immunological and immunofluorescence techniques it was revealed that ceratitins are reabsorbed by the fat body and a fraction of them is deposited in the cuticle. The possible role of ceratitins is discussed.     

Embryonic development of the bovine pineal gland (Bos taurus) during prenatal life (30 to 135 days of gestation)

The ontogenesis of the pineal gland of 30 bovine embryos (Bos taurus) has been analysed from 30 until 135 days of gestation by means of optical microscopy and immunohistochemical techniques. For this study, the specimens were grouped into three stages in accordance with the most relevant histological characteristics: Stage 1 (30 to 64 days of prenatal development); Stage 2 (70 to 90 days) and Stage 3 (106 to 135 days). In the cow, it is from 30 days of gestation that the first glandular outline becomes differentiated from the diencephalic ependyma of the third ventricle. This differentiation includes the phenomena of proliferation and multiplication of the ependymal cells that form the epithelium of the pineal outline in development. At 82 days of intrauterine life, in the interior of the pineal parenchyma, we witnessed some incipient pseudoglandular structures that at 135 days were well differentiated. The pineal parenchyma displays a cytology made up of two cellular types of structurally distinct characteristics: pinealoblasts and interstitial cells. Both cellular types begin differentiation at 70 days of embryonic development, the pinealoblasts being greater in number than the interstitial cells. The glandular stroma is formed from the capsular, trabecular and the perivascular connective tissue, filling the interparenchymal space. A dense network of capillaries, which drive across the trabecular connective tissue towards the central glandular zone where their density increases and their calibre is reduced, complete the glandular structure. GFAP positive cells were observed in the embryonic pineal parenchyma in stage 3. At 135 days of gestation, NPY positive fibers entered the pineal gland through the pineal capsule occupying a perivascular localization. Morphological studies of this nature are vital for future use as parameters, indicative of the functional activity of the bovine pineal gland during embryonic development.     

Expression of hindlimb abnormalities under rearing temperature effects during the larval development of the salamander Pleurodeles waltl (urodele amphibian)

Anomalie m.p. is a spontaneous and heritable hindlimb abnormality described earlier. Twenty years later, Pleurodeles waltl larvae from the strain bearing anomalie m.p. and reared at room temperature or at 30°C, expressed abnormalities (ectrodactylia, hemimelia, ectromelia). A morphological study of all the hindlimbs and an analysis of the hindlimb skeleton of samples from the experimental animals confirmed that most of the skeletal malformations were identical to those previously reported and affected the disto-proximal and prepostaxial pattern of the hindlimb. Analysis of the effects of rearing temperature on the expression of anomalie m.p. showed that the effects varied according to the developmental period at which the heat treatment was applied; the sooner the heat treatment began, the more numerous and more various were the degrees of severity of the malformations. Moreover, heat treatment induced the expression of two additional malformations not yet described: the first one, named 'reversed knee joint', was characterized by a reversal of the knee joint, and the second one, named 'twisted foot', by a downward twisting of the foot. The epigenetic effects of rearing at 30°C on hindlimb development are discussed with regard to the differentiation or patterning.     

Ovarian steroid metabolism during post-natal development in the normal mouse and in the adult hypogonadal (hpg) mouse

Ovarian steroid metabolism was investigated (i) during development in a normal inbred strain in which post-natal follicle growth has been described and (ii) in adult hypogonadal (hpg) mice which lack GnRH and have very low serum concentrations of gonadotrophins. Tissue was incubated with [3H]pregnenolone or [3H]androstenedione and metabolites separated by t.l.c. or h.p.l.c. Progesterone was the major metabolite formed at all ages while androstenedione was the major androgen. Between 7 and 21 days there was an overall increase in steroidogenic enzyme activity with a peak of 5 alpha-reductase between 21 and 29 days. The major metabolite of progesterone around puberty was 5 alpha-pregnane-3 alpha-ol-20-one. A sharp increase in 20 alpha-hydroxysteroid dehydrogenase was observed after 38 days due, presumably, to the appearance of corpora lutea. Unlike the rat, androstanediol levels were low at all ages. Oestradiol was the major oestrogen formed from androstenedione with a peak of production at 38 days. In the adult hpg mouse metabolism was similar to that of the 7-day normal mouse although 17-ketosteroid reductase and aromatase levels were very low compared to normal animals of any age, indicating that gonadotrophin stimulation is involved in the expression of activity by these enzymes.     

An immunocytological study of the adult crab-eating macaque (Macaca fascicularis) pituitary and its cytological differentiation during fetal life

The pituitaries of adult and fetal crab-eating macaques (Macaca fascicularis) have been studied by immunofluorescence using 15 antibodies against most of the known hormones in the adenohypophysis. The antibodies used were first checked on adult pituitaries for their cross-specificity with macaque pituitary hormones. We found five types of endocrine cells reacting positively, according to the biochemical relation of the molecules evidenced with one or more of the antibodies used. The sequential appearance of the various hormones in the cells of the anterior and intermediate lobes was then determined. The first hormones evidenced at day 45 of pregnancy were ACTH, beta-MSH, beta- and gamma-LPH and alpha- and beta-endorphins. alpha-MSH appeared at day 48 and STH at day 51. The glycoprotein hormones, LH, FSH and TSH, appeared at day 57 but the thyrotropes and gonadotropes did not attain their adult characteristics (staining intensity, morphology, density and distribution in the pituitary) until days 71 and 93, respectively. Prolactin was only found beginning at day 93 of pregnancy. The different specificity tests applied to the pituitary of the macaque, as well as to that of other vertebrates, show that the antibodies used have good specificity. A comparison of the dates at which the fetal pituitary gonadotropes appear in the macaque and the results of a developmental study of the external genital organs in that species indicated that the pituitary gonadotropic function is only established after somatic sex differentiation, which would thus probably occur independently.