Immunofluorescent Detection of Two Thymidine Analogues (CldU and IdU) in Primary Tissue

Accurate measurement of cell division is a fundamental challenge in experimental biology that becomes increasingly complex when slowly dividing cells are analyzed. Established methods to detect cell division include direct visualization by continuous microscopy in cell culture, dilution of vital dyes such as carboxyfluorescein di-aetate succinimidyl ester (CFSE), immuno-detection of mitogenic antigens such as ki67 or PCNA, and thymidine analogues. Thymidine analogues can be detected by a variety of methods including radio-detection for tritiated thymidine, immuno-detection for bromo-deoxyuridine (BrdU), chloro-deoxyuridine (CldU) and iodo-deoxyuridine (IdU), and chemical detection for ethinyl-deoxyuridine (EdU). We have derived a strategy to detect sequential incorporation of different thymidine analogues (CldU and IdU) into tissues of adult mice. Our method allows investigators to accurately quantify two successive rounds of cell division. By optimizing immunostaining protocols our approach can detect very low dose thymidine analogues administered via the drinking water, safe to administer to mice for prolonged periods of time. Consequently, our technique can be used to detect cell turnover in very long-lived tissues. Optimal immunofluoresent staining results can be achieved in multiple tissue types, including pancreas, skin, gut, liver, adrenal, testis, ovary, thyroid, lymph node, and brain. We have also applied this technique to identify oncogenic transformation within tissues. We have further applied this technique to determine if transit-amplifying cells contribute to growth or renewal of tissues. In this sense, sequential administration of thymidine analogues represents a novel approach for studying the origins and survival of cells involved in tissue homeostasis

Haematopoietic stem cells do not asymmetrically segregate chromosomes or retain BrdU

Stem cells are proposed to segregate chromosomes asymmetrically during self-renewing divisions so that older ('immortal') DNA strands are retained in daughter stem cells whereas newly synthesized strands segregate to differentiating cells(1-6). Stem cells are also proposed to retain DNA labels, such as 5-bromo-2-deoxyuridine (BrdU), either because they segregate chromosomes asymmetrically or because they divide slowly(5,7-9). However, the purity of stem cells among BrdU-label-retaining cells has not been documented in any tissue, and the 'immortal strand hypothesis' has not been tested in a system with definitive stem cell markers. Here we tested these hypotheses in haematopoietic stem cells (HSCs), which can be highly purified using well characterized markers. We administered BrdU to newborn mice, mice treated with cyclophosphamide and granulocyte colony-stimulating factor, and normal adult mice for 4 to 10 days, followed by 70 days without BrdU. In each case, less than 6% of HSCs retained BrdU and less than 0.5% of all BrdU-retaining haematopoietic cells were HSCs, revealing that BrdU has poor specificity and poor sensitivity as an HSC marker. Sequential administration of 5-chloro-2-deoxyuridine and 5-iodo-2-deoxyuridine indicated that all HSCs segregate their chromosomes randomly. Division of individual HSCs in culture revealed no asymmetric segregation of the label. Thus, HSCs cannot be identified on the basis of BrdU-label retention and do not retain older DNA strands during division, indicating that these are not general properties of stem cells.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/62821/1/nature06115.pd

Inducible deletion of Dicer or Drosha reveals multiple functions for miRNAs in postnatal epidermis and hair follicles

miRNAs make up one of the most abundant classes of regulatory molecules as each miRNA is estimated to regulate hundreds of mRNAs. The repressive effects on individual target mRNAs are typically relatively mild and the deletion of individual or even multiple related miRNAs often results in subtle phenotypes. Changes in the miRNA expression profiles of multiple tumor samples, including melenoma and epithelial skin cancers, suggest a widespread alteration in miRNA networks during tumorigenesis. Similarily, miRNA networks are essential for the development of multiple systems, including the hair follicle. Although a few epidermal miRNAs have been studied in adult skin, global miRNA loss has not been evaluated. Two key endonucleases, Dicer and Drosha, are required for miRNA biogenesis; however both enzymes have miRNA-independent functions of processing siRNA or preribosomal RNAs, respectively. To elucidate miRNA function in adult skin, we generated mice with inducible epidermal deletion of each endonuclease and assayed for shared phenotypes. Deletion of Dicer or Drosha in adult life resulted exclusively in a shared spectrum of phenotypes, indicating that their major function is in miRNA biogenesis, rather than in non-miRNA dependent roles. Interestingly neither enzyme was necessary to maintain resting hair follicles, but both were required during distinct phases of adult hair follicle growth: for the viability of the transient amplifying population; for normal hair shaft formation; and for initiation of hair follicle regression. After prolonged loss of either enzyme, follicular degradation occurred concomitantly with epidermal thickening and dermal inflammation, suggesting additional miRNA roles in hair follicle maintenance and epidermal homeostasis. Analysis of miR-205 targets, a miRNA highly expressed throughout the hair follicle growth cycle, revealed significantly increased levels of Zeb2, and E2F1 in Dicer mutant skin suggesting that deregulation of these factors may contribute to Dicer and Drosha mutant phenotypes, facilitating future studies of their regulation and function in the skin. These results demonstrate specific requirements for miRNAs in maintaining the ability of adult hair follicles to grow and regenerate, and indicate that Drosha and Dicer dependent miRNAs play multiple roles at successive time points of the hair follicle growth cycle

FAKTOR-FAKTOR YANG MEMPENGARUHI KOMPETENSI SDM DALAM UPAYA MENINGKATKAN PRODUKTIVITAS KERJA (STUDI KASUS PADA INDUSTRI RAJUTAN MILKAN KNIT)

Tujuan penelitian ini adalah untuk mengetahui faktor-faktor yang mempengaruhi kompetensi SDM dalam meningkatkan produktivitas industri kreatif rajutan Milkan Knit. Untuk mengetahui faktor yang paling dominan mempengaruhi kompetensi SDM dalam meningkatkan produktivitas industri kreatif rajutan Milkan Knit. Untuk mengetahui seberapa besar pengaruh faktor dominan kompetensi SDM dalam meningkatkan produktivitas industri kreatif rajutan Milkan Knit. Metode penelitian yang digunakan dalam penelitian ini yaitu metode penelitian deskriptif dan verifikatif dengan pendekatan explanatory survey. Sampel penelitian ini berjumlah 30 responden. Metode analisa data menggunakan analisis faktor. Hasil penelitian yang diperoleh adalah Faktor-faktor yang mempengaruhi kompetensi SDM dalam meningkatkan produktivitas industri kreatif rajutan Milkan Knit yaitu sebanyak lima komponen yaitu faktor pengetahuan, penerapan teori, inovasi produk, masa kerja, pengalaman kemampuan kerja, dan faktor adaptasi. Faktor yang paling dominan mempengaruhi daya saing SDM dalam meningkatkan produktivitas industri kreatif rajutan Milkan Knit adalah pada komponen pendidikan yaitu pengetahuan yang dimiliki. Besarnya pengaruh faktor dominan daya saing SDM dalam meningkatkan produktivitas industri kreatif rajutan Milkan Knit yaitu 75,9%

Cyclins D2 and D1 Are Essential for Postnatal Pancreatic β-Cell Growth

Regulation of adult β-cell mass in pancreatic islets is essential to preserve sufficient insulin secretion in order to appropriately regulate glucose homeostasis. In many tissues mitogens influence development by stimulating D-type cyclins (D1, D2, or D3) and activating cyclin-dependent kinases (CDK4 or CDK6), which results in progression through the G(1) phase of the cell cycle. Here we show that cyclins D2 and D1 are essential for normal postnatal islet growth. In adult murine islets basal cyclin D2 mRNA expression was easily detected, while cyclin D1 was expressed at lower levels and cyclin D3 was nearly undetectable. Prenatal islet development occurred normally in cyclin D2(−)(/)(−) or cyclin D1(+/)(−) D2(−)(/)(−) mice. However, β-cell proliferation, adult mass, and glucose tolerance were decreased in adult cyclin D2(−)(/)(−) mice, causing glucose intolerance that progressed to diabetes by 12 months of age. Although cyclin D1(+/)(−) mice never developed diabetes, life-threatening diabetes developed in 3-month-old cyclin D1(−)(/+) D2(−)(/)(−) mice as β-cell mass decreased after birth. Thus, cyclins D2 and D1 were essential for β-cell expansion in adult mice. Strategies to tightly regulate D-type cyclin activity in β cells could prevent or cure diabetes

Cyclin D2 Protein Stability Is Regulated in Pancreatic β-Cells

The molecular determinants of β-cell mass expansion remain poorly understood. Cyclin D2 is the major D-type cyclin expressed in β-cells, essential for adult β-cell growth. We hypothesized that cyclin D2 could be actively regulated in β-cells, which could allow mitogenic stimuli to influence β-cell expansion. Cyclin D2 protein was sharply increased after partial pancreatectomy, but cyclin D2 mRNA was unchanged, suggesting posttranscriptional regulatory mechanisms influence cyclin D2 expression in β-cells. Consistent with this hypothesis, cyclin D2 protein stability is powerfully regulated in fibroblasts. Threonine 280 of cyclin D2 is phosphorylated, and this residue critically limits D2 stability. We derived transgenic (tg) mice with threonine 280 of cyclin D2 mutated to alanine (T280A) or wild-type cyclin D2 under the control of the insulin promoter. Cyclin D2 T280A protein was expressed at much higher levels than wild-type cyclin D2 protein in β-cells, despite equivalent expression of tg mRNAs. Cyclin D2 T280A tg mice exhibited a constitutively nuclear cyclin D2 localization in β-cells, and increased cyclin D2 stability in islets. Interestingly, threonine 280-mutant cyclin D2 tg mice had greatly reduced β-cell apoptosis, with suppressed expression of proapoptotic genes. Suppressed β-cell apoptosis in threonine 280-mutant cyclin D2 tg mice resulted in greatly increased β-cell area in aged mice. Taken together, these data indicate that cyclin D2 is regulated by protein stability in pancreatic β-cells, that signals that act upon threonine 280 limit cyclin D2 stability in β-cells, and that threonine 280-mutant cyclin D2 overexpression prolongs β-cell survival and augments β-cell mass expansion