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Biomedical subjects

R Wanke

Publications and source records attributed to R Wanke.

At least 19 recordsLinked to original sources
PubMed2004

Insulin-like growth factor-binding protein-4 inhibits growth of the thymus in transgenic mice.

Numerous in vitro studies have demonstrated that IGF-binding protein (IGFBP)-4 is a consistent inhibitor of IGF actions. In order to investigate the functions of IGFBP-4 in vivo, transgenic mice were generated by microinjection of a transgene, in which the murine Igfbp4 cDNA is driven by the H-2K(b) promoter, and followed by a splicing cassette and polyadenylation signal of the human beta-globin gene. Transgene mRNA was expressed ubiquitously, and elevated IGFBP-4 protein was detected in the spleen, thymus, kidney and lung of transgenic mice. The activities of serum IGFBPs were not changed in transgenic mice. Immunohistochemical studies revealed transgene expression predominantly in the thymic medulla and red pulp of the spleen. Body weight and the weights of the spleen, kidney and lung of transgenic mice were not different from controls. In contrast, the thymus of transgenic mice showed a significantly reduced weight and cortex volume. In transgenic thymus and spleen, cell proliferation was inhibited and apoptosis was stimulated. Transgenic mice showed normal T- and B-cell development and normal basal plasma immunoglobulin levels. In conclusion, overexpression of IGFBP-4 inhibits growth of the thymus. IGFBP-4 excess inhibits cell proliferation and stimulates apoptosis in lymphoid tissues, but does not affect lymphocyte development. These findings suggest that IGFBP-4 is a potential growth inhibitor of lymphoid tissues.

Animals↗
PubMed2001

Nuclear transfer in cattle with non-transfected and transfected fetal or cloned transgenic fetal and postnatal fibroblasts.

The efficiency of nuclear transfer (NT) using two primary cultures of fetal fibroblasts (FF1 and FF2) was compared vs. the same cultures transfected with an expression vector in which the bovine prochymosin coding sequence is placed under the control of the bovine alpha(S1)-casein promoter (TFF1 and TFF2). In addition, fibroblasts of a cloned transgenic fetus (TRFF1) derived from TFF1 and ear skin fibroblasts of a 1-month-old cloned transgenic calf (TRCF1) derived from TRFF1 were used as nuclear donors. Embryos reconstructed from FF1 (44%) and FF2 (52%) developed to the blastocyst stage at a significantly (P < 0.05) higher rate than those derived from TFF1 (24%) and TFF2 (27%). The proportions of cleaved embryos and blastocysts were significantly (P < 0.05) higher with TRFF1 than with TRCF1 used as nuclear donors (75 vs. 66% and 33 vs. 16%, respectively). Transfer of NT embryos derived from FF2 and TFF2 to recipients resulted in similar pregnancy rates on day 30 (52 and 48%, respectively). However, with TFF2 embryos, the majority of pregnancies (8/11; 73%) was lost in the first and second trimesters of gestation, whereas 4/11 (36%) pregnancies with FF2 embryos were lost during the full period of in vivo development. Of 11 FF2 and 6 TFF2 born calves (25 and 13% of transferred embryos, respectively), 6 and 3 survived including one oversized FF2 calf. After transfer of TRFF1 and TRCF1 NT embryos to recipients, initial pregnancy rate was as a tendency higher in the TRFF1 (49%) than in the TRCF1 group (30%). The majority (14/17) of TRFF1 pregnancies and all TRCF1 pregnancies were lost in the first and second trimester. A high proportion of TRFF1 calves (5/8) showed increased body weights, and only two calves which were also large survived. These findings demonstrate that (i) extended culture associated with transfection and selection procedures may induce changes of donor cells which markedly decrease the efficiency of nuclear transfer and (ii) these changes are not reversed by recloning.

Animals↗
PubMed2001

Growth inhibition in giant growth hormone transgenic mice by overexpression of insulin-like growth factor-binding protein-2.

To clarify the role of insulin-like growth factor (IGF)-binding protein-2 (IGFBP-2) in postnatal growth regulation, we crossed hemizygous CMV-IGFBP-2 transgenic mice with hemizygous PEPCK-bGH transgenic mice, which are characterized by serum GH levels in the range of 2 microgram/ml. Four genetic groups were obtained: animals carrying both transgenes (GB), the GH (G) or the IGFBP-2 transgene (B), and nontransgenic controls (C). Male offspring were analyzed for serum levels of IGF-I, for serum and tissue levels of IGFBP-2, and for body and organ growth. Serum IGF-I levels were 2- to 3-fold increased (P < 0.001) in the GH-overexpressing groups, with no difference between G and GB mice. Serum IGFBP-2 levels were 4- to 9-fold (P < 0.001) increased both in B and GB vs. C and G mice. Western immunoblot analysis did not reveal differences in tissue IGFBP-2 levels between B and GB mice. IGFBP-2 levels were highest in pancreas, followed by skeletal muscle, heart, kidney, brain, skin, and spleen. No elevation of IGFBP-2 was found in the liver. Body weight gain of G and GB mice was significantly increased vs. C and B mice, resulting in almost 2-fold increased body weights at the age of 15 weeks. However, there was a significant reduction in body weight of GB vs. G mice (17%; P < 0.001) and of B vs. C mice (13%; P < 0.05). This was primarily caused by a marked reduction of carcass weight (GB vs. G, 27%; B vs. C, 21%; P < 0.001). Measurements of nose-rump-length, organ (brain, heart, spleen, liver, pancreas, kidney), and tissue weights (skin, carcass, abdominal fat) in 5- and 15-week-old mice revealed several indications that the growth-inhibiting effect of IGFBP-2 overexpression was more marked in high-GH/IGF-I mice: 1) At 5 weeks of age, GB mice displayed a significant reduction of all growth parameters except for the weight of abdominal fat, when compared with G mice, whereas only brain weight was significantly reduced in B vs. C mice. 2) In 15-week-old animals, a significant reduction in all growth parameters, except for spleen and abdominal fat weights, was seen in GB vs. G mice, whereas only nose-rump-length and the weights of carcass and brain were significantly reduced in B vs. C mice. Our study demonstrates, for the first time, the potential of IGFBP-2 to inhibit GH-stimulated growth in giant transgenic mice, providing further evidence for an inhibitory effect of this IGFBP in vivo.

Animals↗
PubMed2001

Chronic tubulointerstitial nephropathy in six okapis (Okapia johnstoni).

Renal tubular atrophy with conical and medullary interstitial fibrosis with severe thickening of the basement membranes of atrophic tubules was found in six okapis (Okapia johnstoni). Focal glomerular atrophy, probably secondary to ischemic collapse of the glomerular capillary tuft, was also observed. Although the etiologies and pathogeneses of these nephropathies are unclear, primary damage of the tubular epithelium appears to be the most likely cause, and toxicity from ingested plant material, possibly willow (Salix sp.), is a possibility.

Animals↗
PubMed2001

[Role of podocyte damage in the pathogenesis of glomerulosclerosis and tubulointerstitial lesions: findings in the growth hormone transgenic mouse model of progressive nephropathy].

The sequence of structural changes terminating in glomerulosclerosis, tubular atrophy and interstitial fibrosis was analyzed in the growth hormone (GH) transgenic mouse (TM) model of progressive renal disease. The investigation was performed in TM expressing the bovine GH gene under the control of the murine metallothionein-1-promoter and non-transgenic controls (CM) of different age groups. The kidneys were studied by light microscopy, transmission and scanning electron microscopy, and were analyzed with stereological methods. Early-stage renal lesions were characterized by glomerular hypertrophy and mesangial expansion. In 7-week-old TM the mean glomerular volume was twice that of age-matched CM. The number of endothelial and of mesangial cells per glomerulus was increased in TM vs. CM, while the number of podocytes did not change. The podocytes demonstrated hypertrophy and foot process effacement. Concomitant with an age-related further increase of glomerular size in TM, severe maladaptive podocyte lesions including detachment of podocytes were observed. The resultant denudation of the glomerular basement membrane was associated with severe proteinuria, glomerular hyalinosis, synechia formation and collapse of glomerular capillaries. These lesions progressed to glomerular obsolescence that was associated with atrophy of the adjacent tubule and interstitial fibrosis. The progressive kidney lesions in this model appear to be attributable to a considerable extent to podocyte damage resulting from the limited capacity of this cell type to keep up with progressing overall tuft growth. The findings provide further evidence that mature podocytes are unable for effective cell replication in vivo, and that podocyte damage plays a significant role in the pathogenesis of progressive glomerulosclerosis with tubular atrophy and interstitial fibrosis.

Animals↗
PubMed2000

Effects of IGFBP-2 overexpression in vitro and in vivo.

Insulin-like growth factor-binding proteins (IGFBPs) are important modulators of IGF actions and may have both stimulatory and inhibitory effects. Expression of IGFBP-2 is increased after fasting and in a variety of pathological conditions. However, the specific role of IGFBP-2 in growth physiology remains to be determined. In this review, we summarize data from in vitro and in vivo models suggesting that IGFBP-2 has mainly inhibitory effects on IGF actions. Since the growth hormone (GH)/IGF system is involved in a number of pathological alterations of the kidney and these changes may--at least in part--be due to increased IGF-I, local overexpression of inhibitory IGFBP-2 in the kidney might prevent IGF-I-induced lesions. This hypothesis will be tested by crossing GH transgenic mice, a common model of glomerulosclerosis, with transgenic mice characterized by systemic and renal overexpression of IGFBP-2.

Animals↗
PubMed1999

Adult cloning in cattle: potential of nuclei from a permanent cell line and from primary cultures.

Nuclear transfer was used to evaluate the developmental potential of nuclei from a spontaneously immortalized bovine mammary gland epithelial cell line (MECL) and from primary cultures of mammary gland cells (PMGC) and ear skin fibroblasts (PESF) established from 3-year-old cows. Cell proliferation was investigated by incorporation and detection of 5-bromo-2'-deoxyuridine (BrdU). The proportion of cells in S-phase was significantly (P < 0.05) higher for MECL cells than for PMGC and PESF, both in the presence of serum (90% vs. 28% and 15%) and following serum starvation (27% vs. 6% and 3%). Nuclei from PESF supported the development of reconstructed embryos to the blastocyst stage significantly better than those of PMGC (60% vs. 26%; P < 0.05). Embryos reconstructed with cells from MECL failed to develop to blastocysts. After transfer of embryos derived from PMGC and PESF, respectively, 2/2 and 5/12 recipients were pregnant on day 42. On day 90, the corresponding pregnancy rates were 2/2 and 3/12. One live calf derived from a PMGC was born at day 287 of gestation. Another live PESF-derived calf was delivered by caesarean section at day 286 of gestation. Our study suggests that nuclei from primary cultures of adult cells can be successfully reprogrammed by nuclear transfer, whereas nuclei from a permanent cell line failed to support the development of nuclear transfer embryos.

Animals↗
PubMed1999

Overgrowth of skin in growth hormone transgenic mice depends on the presence of male gonads.

Growth hormone has been shown to possess stimulatory effects on various connective tissues. We observed that skin growth in male rat phosphoenolpyruvate carboxykinase-bovine growth hormone transgenic mice (serum growth hormone levels: 740-1940 ng per ml) is progressive with age, resulting in an "oversized coat" phenotype with a marked increase in absolute and relative skin weight and surface area, and in thickness of the dermis. Histologic changes include severe dermal fibrosis and replacement of subdermal adipose tissue by fibrous tissue. Apart from an increase in skin surface area, these changes were not noted in female transgenic mice, arguing for a specific interaction of growth hormone with male sex hormones. To clarify this point, 6 wk old male transgenic mice and control mice were castrated and compared with their noncastrated counterparts in parameters of skin growth at an age of 8 mo. The skin weight of castrated transgenic mice was smaller (p < 0.01) than that of intact transgenic mice both absolutely and relative to body weight. The relative skin weight of castrated transgenic mice was in the same range as in intact and castrated control mice. Absolute and relative skin area of castrated transgenic mice was greater (p < 0. 001 and p < 0.05) than in controls but lower than in intact transgenic mice (p < 0.001 and p < 0.05). When compared with control mice, intact transgenic mice displayed an increase (p < 0.01) in the thickness of dermis. In castrated transgenic mice the thickness of the dermis was in the same range as in control mice. Our findings demonstrate a specific interaction of growth hormone with male sex hormones resulting in a marked stimulation of skin growth.

Androgens↗
PubMed1999

Overexpression of insulin-like growth factor-binding protein-2 in transgenic mice reduces postnatal body weight gain.

Insulin-like growth factor (IGF)-binding protein-2 (IGFBP-2) has been shown to inhibit IGF-dependent cell proliferation in a number of in vitro studies. However, no in vivo model of IGFBP-2 overexpression has been established so far. Therefore, we have generated transgenic mice, in which expression of a mouse IGFBP-2 complementary DNA is controlled by the cytomegalovirus (CMV) promoter. In two independent transgenic strains, transgene expression was highest in pancreas and stomach, followed by skeletal muscle, heart, colon, spleen, adipose tissue, brain, and kidney. Within the pancreas, IGFBP-2 expression was found in the islets but not in the exocrine part. Serum IGFBP-2 levels of CMV-IGFBP-2 transgenic mice were about 3-fold (P < 0.05) increased, compared with controls, whereas serum levels of IGF-I and IGF-II were unaffected by IGFBP-2 overexpression. Fasted serum glucose and fasted insulin levels were slightly reduced in transgenic mice, compared with controls. Postprandial serum glucose insulin levels were not affected by the genotype. At days later than 23, body weights of transgenic mice were significantly (P < 0.05) reduced in both sexes, compared with nontransgenic littermates. This reduction in body weight was mainly attributable to significantly (P < 0.05) lower carcass weights of CMV-IGFBP-2 transgenic vs. control mice. In contrast, absolute organ weights were not (or only as a tendency) reduced, except for the weight of the spleen, which was significantly (P < 0.05) lower in male transgenic than in control mice. Our data suggest that IGFBP-2 represents a negative regulator of postnatal growth in mice, potentially by reducing the bioavailability of IGF-I.

Animals↗
PubMed1998

Effect of chronic GH overproduction on cardiac ANP expression and circulating ANP levels.

Sodium and water retention are common in acromegaly and upon GH administration. The underlying mechanisms, however, have not been clearly characterized as yet. Therefore, the aim of this study was to examine possible alterations of atrial natriuretic peptide (ANP), an endogenous regulator of volume homeostasis, in response to chronic elevated GH. We used GH-transgenic mice (GH-TM) as a model for chronic hypersomatotropinemia and moreover investigated 7 and 27 week old animals, respectively, in order to discriminate between short and long term effects of GH overexpression. Hematocrit values were reduced in GH-TM compared to control animals and it is known that plasma volume is increased in these animals. Structural lesions of the kidney were found in the GH-TM, however, in the animals studied there were no signs of renal insufficiency as evidenced by serum creatinine and urea levels. The serum concentration of immunoreactive ANP (IR-ANP) determined by RIA was significantly (P < 0.005) elevated in the young GH-TM as compared to control littermates (81.7+/-13.3 vs. 50.9+/-10.8 fmol/ml). The increase in serum IR-ANP of 27 week old GH-TM, however did not reach the level of significance (57.13+/-16.3 vs. 50.25+/-16.4 fmol/ml). Serum samples of control mice as well as of the 7 week old animals mainly contained ANP 99-126, known to be the circulating form of ANP. In contrast, serum of 27 week old GH-TM predominantly showed the cardiac storage form of ANP, ANP 1-126. Cardiac expression of ANP was quantified by Northern blot analysis. mRNA coding for ANP was found 1.2- and 2-fold increased in the atria of 7 and 27 week old GH-TM, respectively. In parallel, a 2.2-fold (7 week) and 2-fold (27 week) increase of IR-ANP was observed in transgenic atria compared to tissue of control animals. In contrast, no significant difference of ANP mRNA expression or of content of IR-ANP was observed in the ventricles of both groups of animals. In conclusion, GH-TM show various alterations in their ANP status suggesting an influence of the peptide on the effect of GH in fluid retention.

Aging↗
PubMed1998

Intestinal dimensions of mice divergently selected for body weight.

BACKGROUND: Selection for body weight provides important animal models for studying mechanisms of growth regulation. This study evaluated growth responses of the gastrointestinal tract (GIT) to long-term selection of mice for high (H line) or low (L line) 8-week body weight as compared with random-bred controls (C line). METHODS: Weights and dimensions of the various parts of the GIT were recorded from 8-month-old mice. For light microscopic stereological analyses, samples were taken from eight equidistant locations covering the whole jejunum/ileum. Vertical sections were used for estimation of fractional volumes of mucosa, submucosa, and muscularis and of villous surface area density and for measurement of villus length. RESULTS: Differences between groups in weights and dimensions of the various parts of the GIT were more pronounced in the proximal than in the distal segments, with greatest values in H, followed by C and L mice. Relative to body weight, intestinal growth was similar in the three lines, except for significantly (P < 0.001) increased relative weights of jejunum/ ileum, caecum, and colon in L mice. The fractional volume of mucosa and villus length decreased, whereas the fractional volumes of submucosa and muscularis increased from the proximal to the distal locations. The absolute volume of mucosa was greatest in H mice, followed by C and L mice. Relative to body weight, the volume of mucosa was significantly (P < 0.01) greater in L mice than in the two other lines. The mean total villous surface area of jejunum/ileum was significantly (P < 0.01) different among the three lines (L line: 144 cm2; C line: 227 cm2; H line: 304 cm2) but proportionate to differences in metabolic body weight. CONCLUSIONS: Selection for body weight affected various parts of the GIT to a different extent. The parameters investigated changed markedly along the small intestine, demonstrating the need for systematic sampling. Vertical section stereology provides unbiased estimates of total villous surface area, which is a parameter of major biological significance.

Animals↗
PubMed1998

Identification of transgenic mice by direct PCR analysis of lysates of epithelial cells obtained from the inner surface of the rectum.

Conventional screening protocols for transgene integration in mice employ tail tips or blood samples as sources to obtain genomic DNA preparations. We have developed a simple alternative non-surgical method. Epithelial cells are scraped off the inner surface of the rectum with a sterile plastic inoculation loop and are lysed with Kawasaki buffer. The lysate can be directly examined in a polymerase chain reaction (PCR) analysis without any need for further DNA purification. This procedure causes minimal harm and stress to the animals and repeated samples can be obtained as often as necessary. This technique has been used successfully to identify transgenic mice from a number of different lines. The method allows quick screening of numerous animals and contributes to a reduction of the number of surgical biopsies required.

Animals↗
PubMed1997

Actions and interactions of growth hormone and insulin-like growth factor-II: body and organ growth of transgenic mice.

To characterize long-term actions and interactions of growth hormone (GH) and insulin-like growth factor-II (IGF-II) on postnatal body and organ growth, hemizygous phosphoenolpyruvate carboxykinase (PEPCK)-human IGF-II transgenic mice were crossed with hemizygous PEPCK-bovine GH transgenic mice. The latter are characterized by two-fold increased serum levels of IGF-I and exhibit markedly increased body, skeletal and organ growth. Four different genetic groups were obtained: mice harbouring the IGF-II transgene (I), the bGH transgene (B), or both transgenes (IB), and non-transgenic controls (C). These groups of mice have previously been studied for circulating IGF-I levels (Wolf et al., 1995a), whereas the present study deals with body and organ growth. Growth curves (week 3 to 12) were estimated by regression with linear and quadratic components of age on body weight and exhibited significantly (p < 0.001) greater linear coefficients in B and IB than in I and C mice. The linear coefficients of male I and C mice were significantly (p < 0.001) greater than those of their female counterparts, whereas this sex-related difference was absent in the bGH transgenic groups. The weights of internal organs as well as the weights of abdominal fat, skin and carcass were recorded from 3.5- to 8-month-old mice. In addition, organ weight-to-body weight-ratios (relative organ weights) were calculated. Except for the weight of abdominal fat, absolute organ weights were as a rule significantly greater in B and IB than in I and C mice. IGF-II overproduction as a tendency increased the weights of kidneys, adrenal glands, pancreas and uterus both in the absence and presence of the bGH transgene. Analysis of relative organ weights demonstrated significant (p < 0.05) effects of elevated IGF-II on the relative growth of kidneys (males and females) and adrenal glands (females), confirming our previous report on organ growth of PEPCK-IGF-II transgenic mice. In females, IGF-II and GH overproduction were additive in stimulating the growth of spleen and uterus, providing evidence for tissue-specific postnatal growth promoting effects by IGF-II in the presence of elevated IGF-I.

Abdomen↗