Flash Labeling Research Articles

Enhancing Worker and Equipment Protection Through Passive Arc-Fault Mitigation

Existing arc flash standards like IEEE 1584 and 1584.1 present models that can be used to estimate incident energy levels in power distribution equipment but provide limited direction regarding how equipment construction should be considered in the calculations. Users performing arc flash risk assessments are left to apply engineering judgment when considering how barriers or isolation of components should be considered. This can lead to risk assessments that are inconsistent or incorrect. Current practices regarding arc flash evaluation in barriered and nonbarriered equipment are reviewed based on current standards. Gaps and potential concerns are discussed, including the lack of defined criteria to use in calculations, along with lack of criteria for determining when barrier systems are considered effective at preventing, mitigating, controlling, or containing arcing faults. A description of a new system that provides passive protection in low-voltage distribution equipment is presented. By providing improved protection for the incoming conductors and main circuit breaker itself, this line-side protection system reduces or eliminates the uncertainties, which allows for more consistent and accurate application of risk assessments. The new protection system enables arc flash protection to be provided by a passive, enclosed system that has been tested to verify performance. As such, the calculations and risk assessment are less reliant on assumptions regarding the performance of the equipment. Practical aspects of arc flash analysis and labeling and operation and maintenance of such equipment are also discussed.

Engineering Extracellular Expression Systems in Escherichia coli Based on Transcriptome Analysis and Cell Growth State

Escherichia coli extracellular expression systems have a number of advantages over other systems, such as lower pyrogen levels and a simple purification process. Various approaches, such as the generation of leaky mutants via chromosomal engineering, have been explored for this expression system. However, extracellular protein yields in leaky mutants are relatively low compared to that in intracellular expression systems and therefore need to be improved. In this work, we describe the construction, characterization, and mechanism of enhanced extracellular expression in Escherichia coli. On the basis of the localizations, functions, and transcription levels of cell envelope proteins, we systematically elucidated the effects of multiple gene deletions on cell growth and extracellular expression using modified CRISPR/Cas9-based genome editing and a FlAsH labeling assay. High extracellular yields of heterologous proteins of different sizes were obtained by screening multiple gene mutations. The enhancement of extracellular secretion was associated with the derepression of translation and translocation. This work utilized universal methods in the design of extracellular expression systems for genes not directly associated with protein synthesis that were used to generate strains with higher protein expression capability. We anticipate that extracellular expression systems may help to shed light on the poorly understood aspects of these secretion processes as well as to further assist in the construction of engineered prokaryotic cells for efficient extracellular production of heterologous proteins.

The novel mitochondria localization of influenza A virus NS1 visualized by FlAsH labeling.

The nonstructural protein 1 (NS1) of the influenza A virus (IAV) is a multifunctional protein that counteracts host cell antiviral responses and inhibits host cell pre‐mRNA processing. NS1 contains two nuclear localization signals that facilitate NS1 shuttling between cytoplasm and nucleus. In this study, we initially observed the novel mitochondria localization of NS1 in a subset of transfected cells. We then further monitored the localization dynamics of the NS1 protein in live cells infected with IAV expressing NS1 with insertion of a tetracysteine‐tag. The resulting mutant virus showed similar levels of infectivity and expression pattern of NS1 to those of wild‐type IAV. Pulse labeling using a biarsenical compound (fluorescein arsenical hairpin binder) allowed us to visualize the dynamic subcellular distribution of NS1 real time. We detected NS1 in mitochondria at a very early infection time point [1.5 h postinfection (hpi)] and observed the formation of a granular structure pattern in the nucleus at 4 hpi. This is the first identification of the novel mitochondria localization of NS1. The possible role of NS1 at an early infection time point is discussed.

Spectroscopic System for Characterization of Human β-Cardiac Myosin

We designed a system to examine the structural properties of human β-cardiac myosin using fluorescence spectroscopy. Expression of human β-cardiac myosin subfragment 1 (M2B-S1) was achieved through the generation of recombinant adenovirus with subsequent infection in the C2C12 embryonic muscle cell line. A tetracysteine site, which enables site-specific labeling of the fluorescein biarscenical hairpin-binding dye (FlAsH), was introduced in the upper 50 kDa (U50) region (residues 316-321) corresponding to the previously utilized site in myosin V. Key conformational changes in the myosin motor, including those associated with nucleotide and actin binding, can be examined by fluorescence resonance energy transfer (FRET) using M2B-S1 FlAsH and fluorescent nucleotides or IAEDANs-labeled actin. We demonstrate the enzymatic properties of M2B-S1 FlAsH are similar to unlabeled M2B-S1, and both are capable of moving actin filaments in the in vitro motility assay. We utilized FRET between M2B-S1 FlAsH and Cy3ATP to examine structural changes in the U50 domain. The kinetics of structural changes were examined during the ATP-binding and actin-activated product release steps. We also designed a system to study structural changes in the force-generating lever arm using FlAsH labeling at the N-terminus of a human β-cardiac myosin construct containing a C-terminal GFP (M2B-GFP). The spectroscopic approach can be used as a tool to study the effects of hypertrophic cardiomyopathy (HCM) mutations on myosin structure or to study the impact of drugs that alter myosin motor activity.

Abstract 1381: A high-throughput assay for tumor endothelial marker-8 (TEM8/ANTXR1) inhibitors

Abstract TEM8 was first described as a gene upregulated in colorectal tumor endothelium compared to quiescent endothelium. It is also a receptor for anthrax toxin. It is expressed in the endothelium of bladder, esophageal and lung tumors. Mouse knockout studies have shown that TEM8 expression, within the host, promotes tumor growth. Endothelial TEM8 expression can control cell shape, migration, and adhesion to extracellular matrix (ECM) proteins in vitro, supporting a role for TEM8 in tumor angiogenesis. TEM8 interacts with collagen I, collagen VI and gelatin. Protective antigen is the component of anthrax toxin that binds to its cell surface receptors. We have demonstrated that a mutated form of protective antigen (PASSSR) inhibits endothelial cell migration, corneal angiogenesis, and tumor growth. We hypothesize that PASSSR binds to TEM8 and inhibits its interaction with ECM proteins, thereby inhibiting angiogenesis and tumor growth. As a large immunogenic protein, PASSSR would have significant challenges as a potential therapeutic molecule. Thus our objective is to establish a high-throughput assay to identify small molecule inhibitors of TEM8, which can serve as lead molecules in the identification of novel antiangiogenic agents. Fluorescence resonance energy transfer (FRET) is a sensitive method well suited to high-throughput screening. We successfully labeled recombinant PAE733C with AlexaFluor546 maleimide. However, identifying a TEM8 labeling strategy proved more challenging. Maleimide or FlAsH labeling of native protein TEM8 extracellular domain, truncations, point mutations or tetracysteine motif extensions resulted in insoluble or inactive protein, as did NHS labeling. We thus investigated GFP labeling, and found that the resulting protein was stable and interacted with PA in the expected manner. When formatted for high-throughput screening, the PA*546 and TEM8-GFP FRET pair gave a Z’ score (a measure of assay performance) of >0.8. We discovered that alterations to the TEM8 molecule, that disrupt either of two pairs of disulfide bonds on cysteine residues, prevent its ability to interact with PASSSR. The FRET assay was used to identify inhibitors, from a library of over 5,000 known bioactive compounds. Strong hits (>50% inhibition of the PASSSR/TEM8 interaction) included ebselen, and phenylmercuricacetate. Ebselen, a selenium-containing glutathione peroxidase mimic, is known to disrupt disulfide bonds and is likely to have inhibited FRET via binding important cysteine containing residues of TEM8. We will assess anti-angiogenic effects of identified known bioactive molecules. We expect that screening larger small molecule libraries of over 250,000 compounds available at ICCB, Longwood, HMS will result in the identification of a number of candidate TEM8 inhibitors. These molecules are likely to have anti-angiogenic and anti-tumorigenic activities. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1381. doi:10.1158/1538-7445.AM2011-1381

Rotational Diffusion of the α2a Adrenergic Receptor Revealed by FlAsH Labeling in Living Cells

The fluorescein arsenical hairpin binder (FlAsH) shows much promise to determine the relative orientations of protein regions and structures even in living cells and in the plasma membrane. In this study, we characterized FlAsH's photophysical properties by steady-state anisotropy and time-resolved single photon counting for further applications with G-protein coupled receptors. We find that FlAsH has a relatively high initial anisotropy of 0.31 ± 0.01 and a three-component fluorescence lifetime with an average of 4.1 ± 0.1 ns. We characterized the FlAsH fluorophore orientation in the α 2A adrenergic receptor revealing rigid orientations of FlAsH in the membrane plane for rotational correlation times of ∼50 ns in living cells. To elucidate the fluorophore-membrane orientation and rotational correlation time, an anisotropy treatment similar to that of another researcher (Axelrod, D. 1979. Biophys. J. 26:557–573) was developed. The rotational correlation times were observed to increase by up to 16 ns after agonist addition. The rotational correlation time also allowed for a comparison to the theoretical relationship between translational and rotational diffusion (originally proposed by Saffman, P. G., and M. Delbrück. 1975. Proc. Natl. Acad. Sci. USA. 72:3111–3113) and revealed a discrepancy of a factor between 10 and 100.

Cell proliferative activity in the kidney of young growing rat analyzed using flash and cumulative labeling with bromodeoxyuridine

We examined the cell proliferation activity of kidney in young growing rats using flash and cumulative labeling with bromodeoxyuridine (BrdU). Rats were subjected to the study at the age of 6 weeks, and cumulative labeling was carried out for periods of 7 to 28 days. BrdU-positive cells were observed after flash labeling and were increased by cumulative labeling. The positive epithelia were mainly distributed in the cortex and the outer stripe of the outer medulla and were scarce in the inner stripe of the outer medulla and inner medulla throughout all labeling periods. In the tubular epithelium, the majority of positive cells were found in the proximal tubule. In the proximal tubule, positive epithelia were abundant in the medullary rays and in the outer stripe of the outer medulla. In the intermediate tubule to collecting duct, positive epithelia were rare. In the renal corpuscle, positive nuclei were mainly found in the endothelial cells and the mesangial cells and were scarce in the parietal cells of the Bowman's capsule. BrdU-positive nuclei were not observed in podocytes. These results indicate that renal tubules actively grow relative to epithelial proliferation, and that the endothelial cells, the mesangial cells and the parietal cells in the renal corpuscle also proliferate at the age of 6 to 10 weeks in rats. For assessment of renal toxicity using young growing rats, not only the morphologic and physiologic features unique to the kidney but also the growing process of the kidney should be taken into account.

Novel Method for Investigation of Interactions between Mitochondrial Creatine Kinase and Adenine Nucleotide Translocase

The aim of this study was to elaborate fluorescent labeling of mitochondrial creatine kinase (MtCK) and adenine nucleotide translocase (ANT) to investigate the mechanism of their functional coupling with Forster resonance energy transfer (FRET) technique. New alternative fluorescent labeling technique - Fluorescein Arsenical Hairpin (Flash/tetracystein) binder technology was exploited to fluorescently label MtCK. Implementation of fluorescent proteins such as GFP for MtCK fluorescent tagging was excluded because of the functional importance of MtCK C- and N-terminal part and insertion of large fluorescent protein inside the MtCK protein imposes potential risk to interfere the structure, localization and function of the fused protein. Tetracysteine motifs were introduced into five different positions in MtCK by mutagenesis. Sequentially the recombinant MtCK constructs were expressed in different eukaryotic cells lines and activity of the constructs were determined. The cells were stained with Flash labeling reagent and the expression of tetracysteine tagged MtCK mutants were visualized ab inito with epifluorescent and confocal microscopy. Improved variant of cyan fluroescent protein Cerulean as an appropriate FRET partner for Flash was chosen to fluorescently label ANT. Both N- and C-terminally fused ANT-Cerulean constructs were generated. ANT fusion proteins were expressed in different eukaryotic cell lines and their expression was visualized with epifluorescent and confocal microscopy. Functional constructs of MtCK and ANT-Cerulean were selected for studies of their interaction in cardiomyocytes by applying FRET technique.

Clonal evolution and progression of 20-methylcholanthrene-induced squamous cell carcinoma of mouse epidermis as revealed by DNA instability and other malignancy markers.

We examined the clonal evolution of skin malignant lesions by repeated topical applications of 20-methylcholanthrene (20-MC) to the skin, which induces hyperplastic epidermis, papillomatous lesion and invasive carcinoma in mice. The lesions were examined histologically and immunohistochemically with anti-single-stranded DNA after acid hydrolysis (DNA-instability test), p53, VEGF, DFF45, PCNA and AgNORs parameters analyses. Multiple clones with increased DNA instability comparable to that of invasive carcinoma were noted in early-stage (2-6 weeks) hyperplastic epidermis, and their number increased in middle (7-11 weeks), and late-stages (12-25 weeks) of hyperplastic epidermis, indicating that they belong to the malignancy category. All papillomatous lesions and invasive carcinomas showed a positive DNA-instability test. Positive immunostaining for various biomarkers and AgNORs parameters appeared in clones with a positive DNA-instability test in early-or middle-stage hyperplastic epidermis, and markedly increased in late-stage hyperplastic epidermis, papillomatous lesions and invasive carcinomas. The percentage of PCNA-positive vascular endothelial cells was significantly higher in VEGF-positive lesions with a positive DNA-instability test and became higher toward the late-stage of progression. Cut-woundings were made to papillomatous and invasive carcinoma lesions, and the regeneration activity of vascular endothelial cells was determined by using flash labeling with tritiated thymidine (3H-TdR). In small papillomatous lesions, vascular endothelial cells showed regenerative response, but the response was weak in large lesions. No such response was noted in invasive carcinomas; rather, cut-wounding induced collapse of blood vessels, which in turn induced massive coagulative necrosis of cancer cells. These responses can be interpreted to reflect exhausted vascular growth activity due to excessive stimulation by VEGF-overexpression, which was persistently seen from hyperplastic epidermis to invasive carcinoma.

Photochemical control of FlAsH labeling of proteins

Spatiotemporal control of protein fluorescence is a powerful tool in tracking protein movements within cells. Here we report an approach to using genetically encoded photo-caged amino acids to control labeling protein tetracysteine tags with biarsenical fluorescein dyes (FlAsH).

Introducing tetraCys motifs at two different sites results in a functional dopamine transporter

We have introduced tetracysteine motifs into different positions of the dopamine transporter (DAT) for specific FlAsH labeling. Two of the constructs expressed at the cell surface and were functional as determined by [3H] dopamine uptake experiments. The N-terminally modified transporter showed uptake levels comparable to the wild-type DAT, while the construct with tetracysteine motif at position 511 displayed an uptake level about 1/3 of its wild-type counterpart. In addition, these two transporter constructs were visualized on the cell surface following labeling with a fluorescent cocaine analog. YFP introduced into the same N-terminal position was also shown to have surface staining in agreement with activity tests. We propose that these two sites are suitable targets for tetracysteine labeling to be used in FlAsH staining studies, while p134, p342, p427, p433 and p517 sites are not.

Conformational detection of prion protein with biarsenical labeling and FlAsH fluorescence

Prion diseases are associated with the misfolding of the host-encoded cellular prion protein (PrP C) into a disease associated form (PrP Sc). Recombinant PrP can be refolded into either an α-helical rich conformation (α-PrP) resembling PrP C or a β-sheet rich, protease resistant form similar to PrP Sc. Here, we generated tetracysteine tagged recombinant PrP, folded this into α- or β-PrP and determined the levels of FlAsH fluorescence. Insertion of the tetracysteine tag at three different sites within the 91–111 epitope readily distinguished β-PrP from α-PrP upon FlAsH labeling. Labelling of tetracysteine tagged PrP in the α-helical form showed minimal fluorescence, whereas labeling of tagged PrP in the β-sheet form showed high fluorescence indicating that this region is exposed upon conversion. This highlights a region of PrP that can be implicated in the development of diagnostics and is a novel, protease free mechanism for distinguishing PrP Sc from PrP C. This technique may also be applied to any protein that undergoes conformational change and/or misfolding such as those involved in other neurodegenerative disorders including Alzheimer’s, Huntington’s and Parkinson’s diseases.

From the test tube to the cell: Exploring the folding and aggregation of a β‐clam protein

A crucial challenge in present biomedical research is the elucidation of how fundamental processes like protein folding and aggregation occur in the complex environment of the cell. Many new physico-chemical factors like crowding and confinement must be considered, and immense technical hurdles must be overcome in order to explore these processes in vivo. Understanding protein misfolding and aggregation diseases and developing therapeutic strategies to these diseases demand that we gain mechanistic insight into behaviors and misbehaviors of proteins as they fold in vivo. We have developed a fluorescence approach using FlAsH labeling to study the thermodynamics of folding of a model beta-rich protein, cellular retinoic acid binding protein (CRABP) in Escherichia coli cells. The labeling approach has also enabled us to follow aggregation of a modified version of CRABP and chimeras between CRABP and huntingtin exon 1 with its glutamine repeat tract. In this article, we review our recent results using FlAsH labeling to study in-vivo folding and present new observations that hint at fundamental differences between the thermodynamics and kinetics of protein folding in vivo and in vitro.

FlAsH labeling of a nuclear receptor domain (D domain of ultraspiracle) fused to tetracysteine tag

Biarsenical fluorescein compounds feature unique fluorescence characteristics and special binding mechanism to tetracysteine tags with certain structures and these dyes offer a feasible method for site specific labeling of heterologously expressed proteins. We aimed FlAsH fluorescent labeling of tetracysteine fused hinge region of the ultraspiracle from Drosophila melanogaster (DmUSP-D domain) to facilitate functional studies of this receptor domain. A CCPGCC tetracysteine motif was integrated between His6, Gateway attB1, and Flag tags and attached to the N-terminus of the DmUSP-D. The fusion protein was expressed in Esherichia coli and the FlAsH labeling was performed in bacterial extracts, under conditions which are compatible with receptor function. The dye was bound to the tetracysteine tag with high affinity and complex stability and the labeling proved to be specific for the target fusion protein. Results indicate that FlAsH labeling of the internal CCPGCC motif can be a valuable tool for the functional characterisation of any nuclear receptor domains.

A Practical Approach to Arc Flash Hazard Analysis and Reduction

Recent efforts to quantify the dangers associated with potential arc flash hazards rely on overcurrent protection to remove a given fault condition. The effectiveness of various devices is determined by a clearing time related to the maximum available fault current for each system location. As industrial and commercial facilities begin to embrace arc flash labeling procedures and begin to recognize arc flash prevention as a part of a complete safety program, the current method of calculation will allow them to quantify the incident energy (cal/cm/sup 2/) associated with a maximum three-phase fault condition. Most faults produce current magnitudes less than the three-phase maximum. This paper will consider fault current magnitudes less than that of the maximum three-phase condition, and discuss the resulting calculations for incident energy across the range of current magnitudes. Under these additional scenarios, the performance of various overcurrent protection devices will be demonstrated. Associated considerations for design, modeling, and maintenance will be presented.

Transgenically Encoded Protein Photoinactivation (FlAsH-FALI): Acute Inactivation of Synaptotagmin I

We demonstrate a noninvasive technique for protein photoinactivation using a transgenically encoded tag. A tetracysteine motif that binds the membrane-permeable fluorescein derivative 4′,5′-bis(1,3,2-dithioarsolan-2-yl)fluorescein (FlAsH) was engineered into synaptotagmin I (Syt I4C). Neuronally expressed Syt I4C rescues the syt I null mutation, can be visualized after FlAsH labeling, and is normally distributed at the Drosophila neuromuscular synapse. Illumination of FlAsH bound Syt I4C at 488 nm decreases evoked release in seconds demonstrating efficient fluorophore-assisted light inactivation (FlAsH-FALI) of Syt I. The inactivation of Syt I is proportional to the duration of illumination and follows first-order kinetics. In addition, Syt I FlAsH-FALI is specific and does not impair Syt I-independent vesicle fusion. We demonstrate that Syt I is required for a post-docking step during vesicle fusion but does not function to stabilize the docked vesicle state.

Fluorescent labeling of recombinant proteins in living cells with FlAsH

Publisher Summary FlAsH labeling of recombinant proteins for cellular localization studies can be considered an alternative to the popular method using green fluorescent protein (GFP) fusions, with the FlAsH method having some advantages. The size of the fluorescent tag is considerably smaller: bound FlAsH has a molecular weight of less than 600 and the addition of a FlAsH target site can be as small as four introduced cysteines with an appended peptide adding less than 2 kDa. This compares with a molecular weight of 30,000 for GFP, which is more likely to perturb the native structure and function of the tagged protein. Both FlAsH and GFP tagging generate a fluorescent protein with similar brightness. However, multiple FlAsH sites can be introduced into a protein so that considerably brighter labeling aid in detecting low-abundance proteins. A major advantage of the FlAsH system is the comparative ease of chemical modification of FlAsH. Coupled with the synthetic versatility of organic chemistry, this enables the incorporation of functionalities other than fluorescence into targeted proteins or peptides.

Mechanical loading stimulates bone formation by reactivation of bone lining cells in 13-week-old rats.

The bone formation that occurs in response to mechanical stimulation is generally considered to be a means by which bone adapts to changes in its mechanical environment. We have previously shown that the expression of genes for bone matrix proteins is maximal 72 h after a single 5-minute episode of loading of tail vertebrae of 13-week-old female rats, that the predominant increase in mineralization occurs after 3 days, and that the osteogenic response to mechanical stimulation is not dependent on prior bone resorption. We have now investigated the cellular correlates of this osteogenic response. No proliferation was detected, by pulse or flash labeling, in the trabecular bone surface cells of animals killed 1 h to 10 days after the loading episode. Ultrastructural examination revealed that most of the cells covering the trabecular bone surface of control vertebrae were flat bone lining cells. After mechanical stimulation, the trabecular bone surface cells developed ultrastructural features of osteoblastic differentiation and activity, with acquisition of an increasingly cuboidal shape, rounded nuclei, and abundant rough endoplasmic reticulum. Morphometric analysis of the mean cell area, mean nuclear area, and cell and nuclear height showed that they were all maximal 48 h after loading. By 120 h after loading, the appearances of bone surface cells had reverted to those of control vertebrae. Thus, mechanical loading appears to activate lining cells, with a temporal sequence that correlates with bone matrix production.

Application of Flash and Cumulative Labeling with Bromodeoxyuridine for Analysis of Cell Kinetics of Bone Cells and Chondrocytes in Young Growing Rats.

We examined cell kinetics of bone cells and chondrocytes in the femur of young growing rats using flash and cumulative labeling with bromodeoxyuridine (BrdU). As flash labeling, rats received an intravenous injection of BrdU. As cumulative labeling, rats were implanted with osmotic pumps filled with BrdU into the peritoneal cavity. The BrdU-labeled nuclei were immunohistochemically identified in decalcified paraffin-embedded specimens using anti-BrdU antibody. In the articular cartilage, BrdU-positive chondrocytes were observed in the middle of the cartilage by flash labeling, and the positive cells were constantly increased with the cumulative labeling days. In the growth plate, BrdU-positive chondrocytes were observed in the proliferating zone by flash labeling, and almost all chondrocytes were positive for BrdU by cumulative labeling. In the bone tissues, by flash labeling, the undifferentiated mesenchymal cells which seem to be a source of osteoblast were positively stained for BrdU. By cumulative labeling, BrdU-positive osteoblasts, osteocytes, and osteoclasts were observed and these cells were mainly localized in the metaphysis. The results suggest that the technique using the combination of flash and cumulative labeling the BrdU is useful for analysis of cell kinetics of bone cells and chondrocytes, and will be applicable to the analysis of bone and cartilage changes induced by various compounds and observed in animal models of skeletal diseases.

Ki-67 expression during rat liver regeneration after partial hepatectomy

Antibodies to the cell-cycle-associated Ki-67 protein have been widely used for more than a decade as markers of proliferative cells. The prototype antibody Ki-67 reacted only with snap-frozen human tissue, but a novel antibody, MIB-1, was able to detect the Ki-67 antigen in paraffin wax-embedded human tissue. The ability of MIB-5, a novel antibody reactive with the rat equivalent Ki-67 protein, to immunohistochemically detect cycling parenchymal and littoral cells in the regenerating rat liver is reported. Rats underwent a standard two-thirds partial hepatectomy (PH), and groups of three animals were killed at intervals for up to 192 hours after PH. DNA synthesis was monitored by flash labeling with bromodeoxyuridine, and the response was as expected with a significant upsurge in hepatocyte labeling at 16 to 17 hours after PH. On the other hand, MIB-5 labeled a relatively constant percentage of hepatocytes (4%-8%) during the first 16 hours after PH, before a large proportion became labeled, also at 17 hours. The temporal pattern of MIB-5 labeling was similar to that of bromodeoxyuridine labeling, although, as expected, MIB-5 indices were higher. Semiquantification of Ki-67 messenger RNA (mRNA) levels by reverse-transcription polymerase chain reaction showed modest (fourfold to fivefold) increases in abundance during the first 12 hours after PH, but then levels increased dramatically to be at least 15-fold those of intact liver at 36 hours after PH. Much higher than normal levels of Ki-67 mRNA persisted throughout the period of study and even at 96 hours after PH they were still ninefold greater than normal. This study has shown the usefulness of the MIB-5 antibody to monitor proliferation in the rat liver, and furthermore, the pattern of expression of both the mRNA and the protein suggest that the Ki-67 protein, with hitherto unknown function, is more abundant in DNA synthesis and mitosis than in the early or even very late first G1 phase. (Hepatology 1997 Sep;26(3):573-8)