Cell-specific localization of insulin-like growth factor binding protein mRNAs in rat liver

The liver is a major source of circulating insulin-like growth factor I (IGF-I), and it also synthesizes several classes of IGF binding proteins (IGFBPs). Synthesis of IGF-I and IGFBPs is regulated by hormones, growth factors, and cytokines. They are nutritionally regulated and expressed in developmentally specific patterns. To gain insight into cellular regulatory mechanisms that determine hepatic synthesis of IGF-I and IGFBPs and to identify potential target cells for IGF-I within the liver, we studied the cellular sites of synthesis of IGF-I, IGF receptor, growth hormone (GH) receptor, and IGFBPs in freshly isolated rat hepatocytes, endothelial cells, and Kupffer cells. We also localized cellular sites of IGFBP synthesis by in situ hybridization histochemistry. Western ligand and immunoblot analyses were used to determine IGFBP secretion by isolated cells. Two IGF-I mRNA subtypes with different 5' ends (class 1 and class 2) were detected in all isolated liver cell preparations. Type 1 IGF receptor mRNA was detected in endothelial cells, indicating that these cells are a local target for IGF actions in liver. GH receptor was expressed in all cell preparations, consistent with GH regulation of IGF-I and IGFBP synthesis in multiple liver cell types. The IGFBPs expressed striking cell-specific expression. IGFBP-1 was synthesized only in hepatocytes, and IGFBP-3 was expressed in Kupffer and endothelial cells. IGFBP-4 was expressed at high levels in hepatocytes and at low levels in Kupffer and endothelial cells. Cell-specific expression of distinct IGFBPs in the liver provides the potential for cell-specific regulation of hepatic and endocrine actions of IGF-I.     

A Versatile System for USER Cloning-Based Assembly of Expression Vectors for Mammalian Cell Engineering

A new versatile mammalian vector system for protein production, cell biology analyses, and cell factory engineering was developed. The vector system applies the ligation-free uracil-excision based technique – USER cloning – to rapidly construct mammalian expression vectors of multiple DNA fragments and with maximum flexibility, both for choice of vector backbone and cargo. The vector system includes a set of basic vectors and a toolbox containing a multitude of DNA building blocks including promoters, terminators, selectable marker- and reporter genes, and sequences encoding an internal ribosome entry site, cellular localization signals and epitope- and purification tags. Building blocks in the toolbox can be easily combined as they contain defined and tested Flexible Assembly Sequence Tags, FASTs. USER cloning with FASTs allows rapid swaps of gene, promoter or selection marker in existing plasmids and simple construction of vectors encoding proteins, which are fused to fluorescence-, purification-, localization-, or epitope tags. The mammalian expression vector assembly platform currently allows for the assembly of up to seven fragments in a single cloning step with correct directionality and with a cloning efficiency above 90%. The functionality of basic vectors for FAST assembly was tested and validated by transient expression of fluorescent model proteins in CHO, U-2-OS and HEK293 cell lines. In this test, we included many of the most common vector elements for heterologous gene expression in mammalian cells, in addition the system is fully extendable by other users. The vector system is designed to facilitate high-throughput genome-scale studies of mammalian cells, such as the newly sequenced CHO cell lines, through the ability to rapidly generate high-fidelity assembly of customizable gene expression vectors.     

Concordance analysis for QTL detection in dairy cattle: a case study of leg morphology

Background

The present availability of sequence data gives new opportunities to narrow down from QTL (quantitative trait locus) regions to causative mutations. Our objective was to decrease the number of candidate causative mutations in a QTL region. For this, a concordance analysis was applied for a leg conformation trait in dairy cattle. Several QTL were detected for which the QTL status (homozygous or heterozygous for the QTL) was inferred for each individual. Subsequently, the inferred QTL status was used in a concordance analysis to reduce the number of candidate mutations.

Methods

Twenty QTL for rear leg set side view were mapped using Bayes C. Marker effects estimated during QTL mapping were used to infer the QTL status for each individual. Subsequently, polymorphisms present in the QTL regions were extracted from the whole-genome sequences of 71 Holstein bulls. Only polymorphisms for which the status was concordant with the QTL status were kept as candidate causative mutations.

Results

QTL status could be inferred for 15 of the 20 QTL. The number of concordant polymorphisms differed between QTL and depended on the number of QTL statuses that could be inferred and the linkage disequilibrium in the QTL region. For some QTL, the concordance analysis was efficient and narrowed down to a limited number of candidate mutations located in one or two genes, while for other QTL a large number of genes contained concordant polymorphisms.

Conclusions

For regions for which the concordance analysis could be performed, we were able to reduce the number of candidate mutations. For part of the QTL, the concordant analyses narrowed QTL regions down to a limited number of genes, of which some are known for their role in limb or skeletal development in humans and mice. Mutations in these genes are good candidates for QTN (quantitative trait nucleotides) influencing rear leg set side view.     

The fetal circadian rhythm in pregnancies complicated by pregestational diabetes is altered by maternal glycemic control and the morning cortisol concentration

Circadian rhythmicity is fundamental to human physiology, and is present even during fetal life in normal pregnancies. The impact of maternal endocrine disease on the fetal circadian rhythm is not well understood. The present study aimed to determine the fetal circadian rhythm in pregnancies complicated by pregestational diabetes mellitus (PGDM), compare it with a low-risk reference population, and identify the effects of maternal glycemic control and morning cortisol concentrations. Long-term fetal electrocardiogram recordings were made in 40 women with PGDM at 28 and 36 weeks of gestation. Two recordings were made in 18 of the women (45.0%) and one recording was made in 22 (55.0%). The mean fetal heart rate (fHR) and the fHR variation (root mean square of squared differences) were extracted in 1-min epochs, and circadian rhythmicity was detected by cosinor analysis. The study cohort was divided based on HbA1c levels and morning cortisol concentrations. Statistically, significant circadian rhythms in the fHR and the fHR variation were found in 45 (100%) and 44 (95.7%) of the 45 acceptable PGDM recordings, respectively. The rhythms were similar to those of the reference population. However, there was no statistically significant population-mean rhythm in the fHR among PGDM pregnancies at 36 weeks, indicating an increased interindividual variation. The group with higher HbA1c levels (>6.0%) had no significant population-mean fHR rhythm at 28 or 36 weeks, and no significant fHR-variation rhythm at 36 weeks. Similarly, the group with a lower morning cortisol concentration (≤8.8 µg/dl) had no significant population-mean fHR-variation rhythm at 28 and 36 weeks. These findings indicate that individual fetal rhythmicity is present in pregnancies complicated by PGDM. However, suboptimal maternal glycemic control and a lower maternal morning cortisol concentration are associated with a less-well-synchronized circadian system of the fetus.     

Decorin in Human Colon Cancer: Localization In Vivo and Effect on Cancer Cell Behavior In Vitro

Decorin is generally recognized as a tumor suppressing molecule. Nevertheless, although decorin has been shown to be differentially expressed in malignant tissues, it has often remained unclear whether, in addition to non-malignant stromal cells, cancer cells also express it. Here, we first used two publicly available databases to analyze the current information about decorin expression and immunoreactivity in normal and malignant human colorectal tissue samples. The analyses demonstrated that decorin expression and immunoreactivity may vary in cancer cells of human colorectal tissues. Therefore, we next examined decorin expression in normal, premalignant and malignant human colorectal tissues in more detail using both in situ hybridization and immunohistochemistry for decorin. Our results invariably demonstrate that malignant cells within human colorectal cancer tissues are devoid of both decorin mRNA and immunoreactivity. Identical results were obtained for cells of neuroendocrine tumors of human colon. Using RT-qPCR, we showed that human colon cancer cell lines are also decorin negative, in accordance with the above in vivo results. Finally, we demonstrate that decorin transduction of human colon cancer cell lines causes a significant reduction in their colony forming capability. Thus, strategies to develop decorin-based adjuvant therapies for human colorectal malignancies are highly rational.     

Linkage relationships in the bovine MHC region. High recombination frequency between class II subregions

Class II genes of the bovine major histocompatibility complex (MHC) have been investigated by Southern blot analysis using human DNA probes. Previous studies revealed the presence of bovine DO , DQ , DQ , DR and DR genes, and restriction fragment length polymorphisms for each of these genes were documented. In the present study, the presence of three additional class II genes, designated DZ , DY , and DY , are reported. DZ was assumed to correspond to the human DZ gene while the other two were designated DY because their relationship to human class II genes could not be firmly established. The linkage relationships among bovine class II genes and two additional loci, TCP1B and C4, were investigated by family segregation analysis and analysis of linkage disequilibrium. The results clearly indicated that all these loci belong to the same linkage group. This linkage group is divided into two subregions separated by a fairly high recombination frequency. One region includes the C4, DQ , DQ , DR and DR loci and the other one is composed of the DO DY , DY , and TCPIB loci. No recombinant was observed within any of these subregions and there was a strong or fairly strong linkage disequilibrium between loci within groups. In contrast, as many as five recombinants among three different families were detected in the interval between these subregions giving a recombination frequency estimate of 0.17 ± 0.07. The fairly high recombination frequency observed between class 11 genes in cattle is strikingly different from the corresponding recombination estimates in man and mouse. The finding implies either a much larger molecular distance between some of the bovine class II genes or alternatively the presence of a recombinational hot spot in the bovine class II region.     

Triploidy—Observations in 154 Diandric Cases

Hydatidiform moles (HMs) are abnormal human pregnancies with vesicular chorionic villi, imposing two clinical challenges; miscarriage and a risk of gestational trophoblastic neoplasia (GTN). The parental type of most HMs are either diandric diploid (PP) or diandric triploid (PPM). We consecutively collected 154 triploid or near-triploid samples from conceptuses with vesicular chorionic villi. We used analysis of DNA markers and/or methylation sensitive-MLPA and collected data from registries and patients records. We performed whole genome SNP analysis of one case of twinning (PP+PM).In all 154 triploids or near-triploids we found two different paternal contributions to the genome (P1P2M). The ratios between the sex chromosomal constitutions XXX, XXY, and XYY were 5.7: 6.9: 1.0. No cases of GTN were observed. Our results corroborate that all triploid human conceptuses with vesicular chorionic villi have the parental type P1P2M. The sex chromosomal ratios suggest approximately equal frequencies of meiosis I and meiosis II errors with selection against the XYY conceptuses or a combination of dispermy, non-disjunction in meiosis I and meiosis II and selection against XYY conceptuses. Although single cases of GTN after a triploid HM have been reported, the results of this study combined with data from previous prospective studies estimate the risk of GTN after a triploid mole to 0% (95% CI: 0–1,4%).     

The murine receptor for urokinase-type plasminogen activator is primarily expressed in tissues actively undergoing remodeling.

uPAR is a cellular receptor for urokinase plasminogen activator, an enzyme involved in extracellular matrix degradation during processes involving tissue remodeling. We have expressed a recombinant soluble form of murine uPAR and raised rabbit polyclonal antibodies to study the expression of uPAR by immunohistochemistry. The immunohistochemical localization of uPAR was determined in normal mouse organs and in tumors formed by the highly metastatic Lewis lung carcinoma. uPAR immunoreactivity was found in the lungs, kidneys, and spleen, and in endothelial cells in the uterus, urinary bladder, thymus, heart, liver, and testis. No uPAR immunoreactivity was detected in muscle. In general, strong uPAR immunoreactivity was observed in organs undergoing extensive tissue remodeling, as exemplified by trophoblast cells in placenta, and in migrating, but not resting, keratinocytes at the edge of incisional wounds. Staining was not detected in any tissue sections derived from uPAR-deficient mice, thus confirming the specificity of the immunohistochemical staining of uPAR in normal mouse tissues. In Lewis lung carcinoma, uPAR immunoreactivity was found in the tumor cells of the primary tumor and in lung metastases. (J Histochem Cytochem 49:237-246, 2001)     

Comparative effects of Aroclor 1254 (polychlorinated biphenyls) and phenanthrene on glucose uptake by freshwater microbial populations.

The effects of polychlorinated biphenyl (PCB) and phenanthrene stress on glucose uptake by natural microbial populations were examined by the heterotrophic potential technique. Temporal and spatial distributions in glucose uptake velocities were examined for natural samples as well as PCB- and phenanthrene-stressed samples. Statistical analysis indicated significant variability among the various samples. It was demonstrated that the environmental variables contributed significantly to the variability in uptake kinetics. Although general trends indicated a PCB-induced stimulation in uptake velocities, these trends were in part masked by sample variability. Data analysis indicated no statistically significant PCB or phenanthrene effect on either total glucose uptake velocities or the proportion of 14CO2 evolved, as compared to natural unstressed samples.     

Binding of a chaperonin to the folding intermediates of lactate dehydrogenase.

When Bacillus stearothermophilus LDH dimer is incubated with increasing concentrations of the denaturant guanidinium chloride, three distinct unfolded states of the molecule are observed at equilibrium [Smith, C. J., et al. (1991) Biochemistry 30, 1028-1036]. The kinetics of LDH refolding are consistent with an unbranched progression through these states. The Escherichia coli chaperonin, GroEL, binds with high affinity to the completely denatured form and more weakly to the earliest folding intermediate, thus retarding the refolding process. A later structurally defined folding intermediate, corresponding to a molten globule form, is not bound by GroEL; neither is the inactive monomer. The complex between GroEL and denatured LDH is destabilized by the binding of magnesium/ATP (Mg/ATP) or by the nonhydrolyzable analogue adenylyl imidodiphosphate (AMP-PNP). From our initial kinetic data, we propose that GroEL exists in two interconvertible forms, one of which is stabilized by the binding of Mg/ATP but associates weakly with the unfolded protein. The other is destabilized by Mg/ATP and associates strongly with unfolded LDH. The relevance of these findings to the role of GroEL in vivo is discussed.