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Biomedical subjects

C S Rosenfeld

Publications and source records attributed to C S Rosenfeld.

At least 19 recordsLinked to original sources
PubMed2007

Fluorescent in situ hybridization for sex chromosome determination before and after fertilization in mice.

In mice, the relative numbers of male and female pups per litter not only can vary but can probably change over the course of pregnancy in response to numerous environmental and physiological factors. As such, a technique is required to determine gender at several developmental stages. Here we describe a robust and accurate fluorescent in situ hybridization (FISH) procedure for determining chromosomal sex that can be applied with minimal modification to sperm, pre-and post-implantation conceptuses and recovered dead post-natal pups. Sperm was prepared for FISH analysis y using a modified microwave decondensation-denaturation technique. Preimplantation conceptuses (0.5dpc) were cultured to the morula stage before sexing. They were then acid-treated to remove the zona pellucida. Tissue homogenates from postimplantational conceptuses (8.5dpc) and stillborn pups were fixed to pre-etched slides. Specimens were hybridized with identical, commercially available DNA probes for the X (FITC) and Y (Cy3) chromosomes. Sperm ratios met the expected value of 0.5 when determined by using XY FISH. Preimplantation conceptuses pre-treated with pepsin yielded distinct fluorescence of X and Y chromosomes in morulae, whereas microwave decondensation resulted in loss of conceptuses from the slide. Both 4.0 and 8.5dpc conceptuses displayed mean sex ratios of 0.5. Post-natal FISH analysis allowed gender identification of pups that could not be sexed due to developmental abnormalities or partial cannibalism. FISH analysis of sperm and of multiple conceptuses or post-natal tissue provided a cost-effective, accurate alternative to PCR-based sex determination.

Animals↗
PubMed2007

Maternal diet composition alters serum steroid and free fatty acid concentrations and vaginal pH in mice.

We examined the effects of three maternal diets (very high fat (VHF), low fat (LF), and control (Purina 5015)) on serum steroids, free fatty acids (FFA), and vaginal pH in National Institutes of Health Swiss mice. Females were fed (VHF, n = 33; LF, n = 33; 5015, n = 48) from 4 to 16 weeks of age. Following breeding, female serum was collected at 0.5 (pre-implantation, early diestrus) or 8.5 (post-implantation, mid-diestrus) days post-coitus (dpc). The serum concentrations of 17beta-estradiol, testosterone, progesterone, and FFA were analyzed at both collection points, and vaginal pH at 0.5 dpc. Striking differences in steroids and FFA were observed at 0.5 dpc among the groups. Estradiol was higher in the VHF (14.1 +/- 3.0 pg/ml), compared with LF mice (5.2 +/- 2.3 pg/ml; P< or = 0.05). In contrast, 0.5 dpc testosterone was lower in the VHF (10.5 +/- 3.0 pg/ml) versus the LF group (32.7 +/- 8.4 pg/ml; P< or = 0.05). At 8.5 dpc, progesterone was higher in the VHF (89.6 +/- 6.7 ng/ml) versus the 5015 group (60.1 +/- 4.9 ng/ml; P< or = 0.05). VHF mice had higher FFA concentrations at 0.5 dpc (1.0 +/- 0.2 mmol/l) than LF and control mice (0.5 +/- 0.1 and 0.6 +/- 0.1 mmol/l respectively; P< or = 0.05). At 8.5 dpc, VHF females had higher serum FFA (0.8 +/- 0.1 mmol/l) than LF and control females (0.4 +/- 0.1 and 0.6 +/- 0.1 mmol/l; P< or = 0.05). Mean vaginal pH of VHF females (6.41 +/- 0.09) was lower than 5015 females (6.76 +/- 0.10; P< or = 0.05). These diet-induced alterations in serum steroid and FFA concentrations might affect several reproductive processes, including preferential fertilization by one class of sperm over the other and sex bias in pre- and post-implantational embryonic development.

Animals↗
PubMed2003

Opportunities for Trisenox (arsenic trioxide) in the treatment of myelodysplastic syndromes.

Arsenic trioxide (ATO) has a long history of efficacy as an antileukemic agent. However, with the advent of modern therapy, it had been relegated to a historical footnote. In the 1990s, investigators in China reported that ATO was safe and had dramatic efficacy in patients with acute promyelocytic leukemia (APL). Preclinical investigations indicate that the biological targets of this novel drug extend to a variety of malignancies other than APL and include induction of apoptosis, nonterminal differentiation, and suppression of proliferation and angiogenesis. The myelodysplastic syndromes (MDSs) present a particular therapeutic challenge. Ineffective hematopoiesis predominates in patients with low-grade prognostic scores. The survival of those patients with high-grade disease is compromised by a high risk of leukemia transformation. Although a number of therapeutic options have been investigated, none has emerged as being broadly efficacious and having an acceptable toxicity profile. No drug has yet received approval by the Food and Drug Administration for this indication. Biologic features of MDS, which include accelerated apoptotic potential, limited maturation capacity, and medullary neovascularity, create a strong scientific rationale for the investigation of ATO in MDS. This report describes the history and scientific basis for ATO treatment of hematologic malignancies, enumerates the potential benefits of ATO in MDS, and discusses the direction of ongoing trials of this novel antineoplastic agent.

Antineoplastic Agents↗
PubMed2003

Evolution of the interferon tau genes and their promoters, and maternal-trophoblast interactions in control of their expression.

It is well established that the interferon tau (IFN-tau) family of proteins play a major role in preventing the regression of the corpus luteum during early pregnancy in ruminants, such as cattle, sheep and goats, but not in other mammals. These interferons, which are structurally and functionally related to type I interferon, such as IFN-alpha and -omega, arose from a duplication of an IFN-omega gene approximately 36 million years ago. The IFN-tau genes have continued to duplicate since that time and have acquired the ability to be transcribed uniquely in the trophectoderm. Low expression is first detectable at the blastocyst stage, but massive transcriptional upregulation occurs a few days later during the initial stages of conceptus elongation. Expression is finally terminated upon trophectoderm attachment to uterine endometrium. The major promoter element that controls expression is an Ets-2/AP-1 enhancer element. Growth factors and cytokines released by the maternal endometrium that, possibly in response to progesterone, act through Ras and the mitogen-activated protein kinase (MAP-kinase) signal transduction pathway have been implicated in controlling IFN-tau gene transcription by activating Ets-2. This timely expression of IFN-tau is not only required to rescue the corpus luteum of pregnancy but may also be an indicator of conceptus fitness, thereby serving as a critical factor that dictates the continuation of pregnancy in ruminants.

Animals↗
PubMed2001

Expression of the bovine oestrogen receptor-beta (bERbeta) messenger ribonucleic acid (mRNA) during the first ovarian follicular wave and lack of change in the expression of bERbeta mRNA of second wave follicles after LH infusion into cows.

In a previous study, the ERbeta cDNA protein-coding region was utilised to clone bovine ERbeta. The objectives in this study were to examine (1) ERbeta mRNA expression in ovarian follicles throughout the bovine first follicular wave, and (2) effect of LH infusion into cows on bERbeta mRNA expression during the second follicular wave. In experiment 1, heifers (4-5 per time point) were ovariectomized at 12, 24, 36, 48, 60, 72, 84, 96, 144, or 216 h after emergence of the first follicular wave after oestrus. In experiment 2, saline or LH was pulsed hourly (computer-controlled syringe pump) into cows (n = 31; 5-6 per treatment) at wave emergence for 2 or 4 days: wave 1-saline (W1S), wave 2-saline (W2S), or wave 2-LH (25 microg/h; W2LH). Ovaries were removed on day 2 or day 4 after wave emergence. Follicles, 2-19mm in size, were dissected, frozen, and stored at -80 degrees C for in situ hybridisation with two bERbeta cRNA probes. Expression of bERbeta mRNA was localised in granulosa cells of healthy follicles. In experiment 1, bERbeta mRNA expression did not change with time points of the wave showing no association of bERbeta mRNA expression with follicular selection and dominance. However, bERbeta mRNA expression decreased with increase in size of all follicles. Expression of bERbeta mRNA was greater in very small follicles (2-4 mm) than in large (> or = 9 mm) follicles. In experiment 2, expression of bERbeta mRNA in follicles did not differ either between W1S and W2S or between W2S and W2LH. In summary, bERbeta mRNA expression decreased with increasing follicular size. However, neither stage of the wave (selection or dominance), nor pulsatile infusion of LH influenced bERbeta mRNA expression.

Animals↗
PubMed2001

Estrogen receptor- and aromatase-deficient mice provide insight into the roles of estrogen within the ovary and uterus.

Estrogen receptor (ER)- (alpha, beta, and both alpha and beta) and aromatase (Ar) knockout (KO) mice have been created to assess the biological effects of estrogens. This review article discusses the ovarian and uterine phenotypes of these mice. The data obtained have confirmed some older inferences about how the steroid acts, but have also revealed some unexpected aspects of estrogen action. Mol. Reprod. Dev. 59:336-346, 2001.

Animals↗
PubMed2001

Association of clonal T-cell large granular lymphocyte disease and paroxysmal nocturnal haemoglobinuria (PNH): further evidence for a pathogenetic link between T cells, aplastic anaemia and PNH.

There is mounting evidence to suggest that T-cell-mediated suppression of haemopoiesis is a pathogenetic mechanism in three bone marrow failure syndromes: aplastic anaemia (AA), paroxysmal nocturnal haemoglobinuria (PNH) and myelodysplasia (MDS). T-cell microclones can be detected by sensitive polymerase chain reaction (PCR)-based methods in all three disorders. Recently, larger clonal populations of T-cell large granular lymphocytes (T-LGLs) have been observed in some patients with AA and MDS. Here, we report the development of a large clonal T-LGL population in a patient with bona fide PNH. In this patient, we defined part of the sequence of the T-cell receptor (TCR) beta-chain gene, and we have shown that the large T-LGL population emerged from a background of multiple smaller T-cell clones. Thus, T-LGL clones in AA, MDS and PNH probably expand as a result of antigenic stimulation. It is postulated that the antigen driving clonal T-cell proliferations in these disorders exists on haemopoietic stem cells.

Adult↗
PubMed2001

An aspartic proteinase expressed in the yolk sac and neonatal stomach of the mouse.

A murine aspartic proteinase, described herein, is intermediate in amino acid sequence identity between the placentally produced pregnancy-associated glycoproteins (PAGs) and gastric pepsins. While PAGs are secreted products of placental trophoblast tissue of ungulates and most are not believed to function proteolytically, pepsins are digestive enzymes. The cDNA for this aspartic proteinase was amplified by reverse transcription-polymerase chain reaction from RNA extracted from murine placentas and neonatal stomachs. The open reading frame encoded a 387-amino acid polypeptide with a 15-residue signal sequence. The enzyme most resembled pepsinogen F (a protein identified in the stomachs of neonatal rabbits and rats) and PAG-like proteins cloned from equine and feline placentae. In the stomach, both its mRNA and protein were expressed in gastric chief cells of preweaned neonates. Within the placenta, its mRNA was present in both the parietal and visceral yolk sacs. However, the protein was most prevalent in the visceral yolk sac, with little detectable in the parietal yolk sac. The recombinant protein was expressed in Escherichia coli. This protein was capable of self-activation and exhibited proteolytic activity toward casein. The presence of this enzyme in two organs involved in the selective transcellular transport of proteins suggests that it has specialized digestive functions.

Amino Acid Sequence↗
PubMed2001

Intraovarian actions of oestrogen.

Oestrogen regulates several hypothalamic and pituitary hormones, which in turn control ovarian functions. Oestrogen and its metabolites, such as catecholoestrogens, also have direct effects within the ovary. This review examines the roles of oestrogen in regulating ovarian folliculogenesis, ovulation and corpus luteum formation. Oestrogen promotes follicular development, which culminates in ovulation, by potentiating follicular development, granulosa cell expression of gonadotrophin receptors, steroidogenesis, and gap junction formation by granulosa cells, and by inhibiting granulosa cell apoptosis. In addition, oestrogen may be needed for corpus luteum formation and maintenance. Studies on mutant mice that either lack one or both of the known oestrogen receptors or are unable to synthesize oestrogen support some but not all of these prior inferences of the roles of oestrogen within the ovary. Although these transgenic mice have proved useful in determining some of the intraovarian actions of oestrogen, they present confounding problems, including hormonal imbalances, that hinder interpretation. Transgenic mice with conditional or tissue-directed mutations in their oestrogen receptors are needed to dissect the ovarian actions of oestrogen further. In addition, microarray technologies, combined with specific hormone treatment regimens are likely to provide an attractive, alternative approach to using mutant mice in clarifying the direct actions of oestrogen in the ovaries of other species.

Animals↗
PubMed2000

Centrosome-centriole abnormalities are markers for abnormal cell divisions and cancer in the transgenic adenocarcinoma mouse prostate (TRAMP) model.

We utilized the transgenic adenocarcinoma mouse prostate (TRAMP) model to study the formation of abnormal mitosis in malignant tumors of the prostate. The results presented here are focused on centrosome and centriole abnormalities and the implications for abnormal cell divisions, genomic instability, and apoptosis. Centrosomes are microtubule organizing organelles which assemble bipolar spindles in normal cells but can organize mono-, tri-, and multipolar mitoses in tumor cells, as shown here with histology and electron microscopy in TRAMP neoplastic tissue. These abnormalities will cause unequal distribution of chromosomes and can initiate imbalanced cell cycles in which checkpoints for cell cycle control are lost. Neoplastic tissue of the TRAMP model is also characterized by numerous apoptotic cells. This may be the result of multipolar mitoses related to aberrant centrosome formations. Our results also reveal that centrosomes at the poles in mitotic cancer cells contain more than the regular perpendicular pair of centrioles which indicates abnormal distribution of centrioles during separation to the mitotic poles. Abnormalities in the centriole-centrosome complex are also seen during interphase where the complex is either closely associated with the nucleus or loosely dispersed in the cytoplasm. An increase in centriole numbers is observed during interphase, which may be the result of increased centriole duplication. Alternatively, these centrioles may be derived from basal bodies that have accumulated in the cell's cytoplasm, after the loss of cell borders. The supernumerary centrioles may participate in the formation of abnormal mitoses during cell division. These results demonstrate multiple abnormalities in the centrosome-centriole complex during prostate cancer that result in abnormal mitoses and may lead to increases in genomic instability and/or apoptosis.

Adenocarcinoma↗
PubMed2000

Gonadotropin induction of ovulation and corpus luteum formation in young estrogen receptor-alpha knockout mice.

Estrogen receptor-alpha (ERalpha) knockout (ERalphaKO) female mice are infertile. Initially, they exhibit normal follicular development, but by 4-5 wk of age, they begin to develop hemorrhagic ovarian cysts. Follicles in adult ERalphaKO female mice progress to the graafian stage, but there are no corpora lutea (CL). To test whether ERalpha is required for ovarian folliculogenesis, ovulation, and CL formation, eCG and hCG were used to ovulate 3- to 5-wk-old ERalphaKO and wild-type (WT) sibling mice. Gonadotropin administration resulted in ovulation in both ERalphaKO and WT mice. Gonadotropin-treated ERalphaKO females that ovulated produced 7.09 +/- 0.77 oocytes per mouse, whereas gonadotropin-treated WT female mice had 16.17 +/- 0.84 oocytes. Surprisingly, ruptured ERalphaKO ovarian follicles developed into CL that had normal morphology. Gonadotropin-treated ERalphaKO mice had 3-fold higher concentrations of serum progesterone than did control ERalphaKO mice that had been administered saline rather than gonadotropins. Thus, the CL in gonadotropin-treated ERalphaKO mice appeared to be steroidogenically functional. On the basis of these findings, ovarian folliculogenesis, ovulation, and CL formation can occur in the absence of ERalpha, although to a lesser extent than in WT mice.

Animals↗
PubMed2000

The differential fate of mesonephric tubular-derived efferent ductules in estrogen receptor-alpha knockout versus wild-type female mice.

We investigated mesonephric tubular-derived efferent ductules in female wild-type (WT) and estrogen receptor-alpha knockout (ERalphaKO) mice from late fetal to adult life. On gestational day 17, efferent ductules in both fetal WT and ERalphaKO females were well developed and morphologically similar, although one third the size of the male counterpart. Unexpectedly, efferent ductules with a ciliated epithelium were still present on postnatal day 10 in WT and ERalphaKO females. By day 23, however, marked phenotypic differences occurred in efferent ductules of WT and ERbetaKO vs. ERalphaKO female mice. In the latter, efferent ductules became hypertrophied and dilated, whereas only small tubules remained in WT and ERbetaKO adult mice. The serum testosterone concentrations were similar in 21- to 25-day-old ERalphaKO, heterozygous, and WT female mice, suggesting that increased testosterone was not inducing enlargement of efferent ductules in ERalphaKO females. In conclusion, remnants of efferent ductules persisted in normal adult female mice, although these structures were greatly reduced in size compared with efferent ductules in ERalphaKO female mice. The underlying mechanism inducing hypertrophy and dilation of efferent ductules in ERalphaKO females is not clear, but secretory and/or reabsorptive function of female efferent ductules may involve ERalpha.

Animals↗
PubMed2000

Normal development of thymus in male and female mice requires estrogen/estrogen receptor-alpha signaling pathway.

Estrogen receptors (ERs) are expressed in the thymus of both males and females, but their role in thymic development and function is unclear. To determine whether ERalpha plays a role in thymic function of either males or females, we compared thymuses of male and female wild-type (WT) and ERalpha knockout (alphaERKO) mice from birth to adulthood. Although thymic size was similar in both male and female WT and alphaERKO mice at birth (d 0), by postnatal d 5 and at all subsequent ages, both male and female alphaERKO mice had significant (30-55%) reductions in thymic weight. Morphometric analysis revealed a reduction in thymic medullary areas in adult alphaERKO mice compared with age-matched WT controls that paralleled thymic involution. There were changes in relative percentages of CD4+ and CD4+CD8+ T-cells, and large decreases (70-80%) in overall absolute numbers of CD4+ and CD4+CD8+ T-cells. Serum corticosterone and testosterone levels were not different in either neonatal or adult male WT or alphaERKO mice, and serum levels of 17beta-estradiol (E2) were similar in neonatal WT and alphaERKO males, indicating that increases in these thymolytic hormones are not responsible for the decreased thymic weight in alphaERKO males. Additionally, delayed-type hypersensitivity was significantly increased in male alphaERKO mice compared with WT mice. In summary, ERalpha deficiency does not inhibit initial differentiation or fetal thymic development, but the absence of ERalpha results in marked decreases in thymic size in both sexes during the postnatal period. These results are the first direct demonstration that the E2/ERalpha signaling system is necessary for maintenance of normal postnatal function of the female thymus gland. The similar results obtained in males demonstrate a role for the E2/ERalpha signaling system in the male thymus and emphasize that estrogens play a more critical role in the male than previously realized.

Animals↗
PubMed1999

Cloning, sequencing, and localization of bovine estrogen receptor-beta within the ovarian follicle.

The potential role of estrogen receptor-beta (ERbeta) in normal ovarian folliculogenesis and in reproductive disorders such as ovarian follicular cysts has not been well defined. Therefore, we were interested in cloning, sequencing, and localizing ERbeta mRNA and protein within the bovine ovary. Bovine ERbeta (bERbeta) was amplified by reverse transcription-polymerase chain reaction (RT-PCR), then cloned and sequenced. Results showed that the open reading frame of bERbeta cDNA spanned 1584 nucleotides encoding a protein of 527 amino acids. The N-terminal region of bERbeta was found to be 80% homologous to human and mouse ERbeta and 79% homologous to rat ERbeta. Bovine ERbeta DNA-binding domain was 100% homologous to human, mouse, and rat ERbeta sequences. The C-terminal/ligand-binding domain of bERbeta was 89% homologous to human, 86% homologous to mouse, and 88% homologous to rat ERbeta. Human and bovine ERbeta amino acid sequences are similar in that their coding region extended farther 5' than initially reported for the published rat ERbeta sequence. Using in situ hybridization and immunohistochemistry, ERbeta mRNA and protein, respectively, were demonstrated to be present in granulosa cells of antral follicles in various stages of follicular growth. These findings suggest a role for bERbeta in ovarian follicular growth and maturation.

Amino Acid Sequence↗
PubMed1999

Congenital ocular anomalies and ventricular septal defect in a dromedary camel (Camelus dromedarius).

A 5-wk-old female dromedary camel (Camelus dromedarius) was clinically diagnosed with bilateral corneal dermoids, incomplete congenital cataracts, a left persistent hyaloid artery (PHA), and a ventricular septal defect (VSD). The corneal dermoids were removed by lamellar keratectomy, and vision improved in the left eye. Thirteen months after dermoid surgery, the calf was presented for enlargement of the right eye. Glaucoma was confirmed in the right eye, and corneal fibrosis and cataract were noted in the left eye. Persistence of the VSD was confirmed by cardiac ultrasonography. The calf was euthanized, and necropsy findings confirmed VSD. Histopathologic examination revealed bilateral corneal thinning and fibrosis, cataracts with retrolental fibroplasia, and retinal dysplasia. Additional changes in the right globe were anterior segment dysgenesis, ruptured lens capsule, chronic phacoclastic uveitis, and retinal separation. The PHA was confirmed in the left eye.

Animals↗
PubMed1998

Transcription and translation of estrogen receptor-beta in the male reproductive tract of estrogen receptor-alpha knock-out and wild-type mice.

Estrogen receptor-alpha (ERalpha) has been identified in the male reproductive tract, but the role of estrogen in the male has not been well characterized. In vivo mutations in ERalpha genes have demonstrated the necessity for ERalpha-mediated action in male fertility. We asked whether both ERbeta messenger RNA and protein were present in the male reproductive tract of wild-type and ERalpha knock-out (ERalpha KO) mice, and whether ERbeta could compensate for the lack of ERalpha in infertile male ERalpha KO mice. Immunohistochemical localization with both N- and C-terminal anti-ERbeta antibodies demonstrated that ERbeta is present in the Leydig cells of the testes and in the epithelium of both the efferent ductules and the initial segment of the epididymis. RT-PCR amplification was used to confirm ERbeta transcription in these tissues. In conclusion, we observed that ERbeta messenger RNA and protein continue to be expressed in the Leydig cells, elongated spermatids, efferent ductules, and the initial segment of the epididymides of ERalpha KO mice, but the presence of ERbeta is not able to compensate for the absence of ERalpha in male reproductive function.

Animals↗
PubMed1997

Phase II study of roquinimex in myelodysplastic syndrome.

A Phase II clinical trial was undertaken using roquinimex (Linomide) in patients with myelodysplastic syndromes (MDS). Roquinimex is an orally active drug with immunostimulating activities demonstrated in vitro and clinically. Seventeen patients with MDS were enrolled in the study. Eligibility was limited to cytopenic patients with <20% marrow blasts. The drug was given orally twice weekly for 12 weeks with frequent monitoring of clinical, hematologic, and immunologic parameters. An increase in CD8+ and CD56+/CD3- cells was detected by 3 weeks. There was, however, no augmentation of natural killer or lymphokine-activated killer cell activity; progenitor cells were unchanged. Four patients had improvement in neutrophil counts, and two patients had improvement in platelet counts. Despite this improvement, the responses were transient or not maintained after discontinuation of therapy. One patient with RAEB, who was red cell transfusion dependent, experienced a complete remission that has persisted 14 months after completion of therapy. Adverse events developed in >25% of patients and included arthralgia, fever, headache, and myalgia. These side effects led to early withdrawal of therapy in five patients. These findings suggest that roquinimex may be of occasional benefit to patients with myelodysplastic syndromes.

Adjuvants, Immunologic↗