A novel method for the evaluation of proximal tubule epithelial cellular necrosis in the intact rat kidney using ethidium homodimer

Background  

Ethidium homodimer is a cell-membrane impermeant nuclear fluorochrome that has been widely used to identify necrotic cells in culture. Here, we describe a novel technique for evaluating necrosis of epithelial cells in the proximal tubule that involves perfusing ethidium homodimer through the intact rat kidney. As a positive control for inducing necrosis, rats were treated with 3.5, 1.75, 0.87 and 0.43 mg/kg mercuric chloride (Hg²⁺, intraperitoneal), treatments which have previously been shown to rapidly cause dose-dependent necrosis of the proximal tubule. Twenty-four h after the administration of Hg²⁺, ethidium homodimer (5 μM) was perfused through the intact left kidney while the animal was anesthetized. The kidney was then removed, placed in embedding medium, frozen and cryosectioned at a thickness of 5 μm. Sections were permeabilized with -20°C methanol and then stained with 4',6-diamidino-2-phenylindole (DAPI) to label total nuclei. Total cell number was determined from the DAPI staining in random microscopic fields and the number of necrotic cells in the same field was determined by ethidium homodimer labeling.     

Probing disorders of the nervous system using reprogramming approaches

The groundbreaking technologies of induced pluripotency and lineage conversion have generated a genuine opportunity to address fundamental aspects of the diseases that affect the nervous system. These approaches have granted us unrestricted access to the brain and spinal cord of patients and have allowed for the study of disease in the context of human cells, expressing physiological levels of proteins and under each patient's unique genetic constellation. Along with this unprecedented opportunity have come significant challenges, particularly in relation to patient variability, experimental design and data interpretation. Nevertheless, significant progress has been achieved over the past few years both in our ability to create the various neural subtypes that comprise the nervous system and in our efforts to develop cellular models of disease that recapitulate clinical findings identified in patients. In this Review, we present tables listing the various human neural cell types that can be generated and the neurological disease modeling studies that have been reported, describe the current state of the field, highlight important breakthroughs and discuss the next steps and future challenges.     

Identification of trans-dominant HIV-1 rev protein mutants by direct transfer of bacterially produced proteins into human cells.

A synthetic rev gene containing substitutions which introduced unique restriction sites but did not alter the deduced amino acid sequence was used as a vehicle to construct mutations in rev. Insertion or substitution mutations within a domain of Rev resulted in proteins able to inhibit the function of Rev protein in trans. Rev function was monitored in a cell line, HLfB, which contained a rev- mutant provirus. HLfB cells require the presence of rev for virus production, which was conveniently monitored by immunoblot detection of p24gag. Trans-dominant mutants were identified after expression in bacteria and delivery into HLfB cells by protoplast fusion. In addition, the trans-dominant phenotype was verified by expression of the mutant proteins in HLfB cells after cotransfection. These studies define a region between amino acid residues 81 and 88 of rev, in which different mutations result in proteins capable of inhibiting Rev function.     

A moving boundary model of acrosomal elongation

A sperm penetrates an egg by extending a long, actin-filled tube known as the acrosomal process. This simple example of biomotility is one of the most dramatic. In Thyone, a 90 m process can extend in less than 10 s. Experiments have shown that actin monomers stored in the base of the sperm are transported to the growing tip of the acrosomal process where they add to the ends of the existing filaments.The force that drives the elongation of the acrosomal process has not yet been identified although the most frequently discussed candidate is the actin polymerization reaction. Developing what we believe are realistic moving boundary models of diffusion limited actin fiber polymerization, we show that actin filament growth occurs too slowly to drive acrosomal elongation. We thus believe that other forces, such as osmotically driven water flow, must play an important role in causing the elongation. We conjecture that actin polymerization merely follows to give the appropriate shape to the growing structure and to stabilize the structure once water flow ceases.Work partially supported by the United States Department of Energy     

Secondary structure of Tetrahymena thermophilia 5S ribosomal RNA as revealed by enzymatic digestion and microdensitometric analysis.

The secondary structure of [32P] end-labeled 5S rRNA from Tetrahymena thermophilia (strain B) has been investigated using the enzymes S1 nuclease, cobra venom ribonuclease and T2 ribonuclease. The results, analyzed by scanning microdensitometry and illustrated by three-dimensional computer graphics, support the secondary structure model of Curtiss and Vournakis for 5S rRNA. Aberrent mobility of certain RNA fragments on sequencing gels was observed as regions of band compression. These regions are postulated to be caused by stable internal base-pairing. The molecule was probed with T2 RNase in neutral (pH 7.5) and acidic (pH 4.5) buffers and only minor structural differences were revealed. One of the helices was found to be susceptible to enzymatic attack by both the single-strand and double-strand specific enzymes. These observations are evidence for the existence of dynamic structural equilibria in 5S rRNA.     

Cross-activation of the Rex proteins of HTLV-I and BLV and of the Rev protein of HIV-1 and nonreciprocal interactions with their RNA responsive elements

The Rex regulatory proteins of human T-cell leukemia virus type I (HTLV-I) and bovine leukemia virus (BLV), and the Rev protein of human immunodeficiency virus type 1 (HIV-1), promote the cytoplasmic accumulation and translation of viral messenger mRNAs encoding structural proteins. Rev and Rex act through cis-acting elements on the viral RNA; these elements are named Rev- and Rex-responsive elements, or RRE and RXRE, respectively. We show that the Rex proteins of HTLV-I and BLV are interchangeable, but only the Rex protein of HTLV-I can substitute for Rev of HIV-1. Rex of HTLV-I and Rev of HIV-1 appear to act on RRE by similar mechanisms. Rev of HIV-1 does not act on the RXRE of HTLV-I or BLV. The nonreciprocal action of Rev and Rex suggests that these factors interact directly with the cis-acting RNA elements of the two viruses.     

Cloning and functional analysis of multiply spliced mRNA species of human immunodeficiency virus type 1

We have used the polymerase chain reaction technique to clone the small multiply spliced mRNA species produced after infection of human cells by a molecular clone of human immunodeficiency virus type 1 (HIV-1). We identified six Rev-expressing mRNAs, which were generated by the use of two splice acceptors located immediately upstream of the rev AUG. The class of small mRNAs included 12 mRNAs expressing Tat, Rev, and Nef. In addition, HIV-1 produced other multiply spliced mRNAs that used alternative splice sites identified by cloning and sequencing. All of these mRNAs were found in the cytoplasm and should be able to produce additional proteins. The coding capacity of the tat, rev, and nef mRNAs was analyzed by transfection of the cloned cDNAs into human cells. The tat mRNAs produced high levels of Tat, but very low levels of Rev and Nef. All the rev mRNAs expressed high levels of both Rev and Nef and were essential for the production of sufficient amounts of Rev. Therefore, HIV-1 uses both alternatively spliced and bicistronic mRNAs for the production of Tat, Rev, and Nef proteins.     

Activation of tat-defective human immunodeficiency virus by ultraviolet light

Ultraviolet light (UV) is known to cause activation of gene expression from the human immunodeficiency virus type 1 (HIV-1) promoter. To address the question of whether tat-defective HIV-1 provirus could be rescued by UV irradiation we examined its effect on HeLa cells containing integrated proviruses with tat mutations. Exposure of these cells to an optimal dose of UV resulted in the production of infectious viruses. The degree of UV activation and reversion to infectious virus appeared to depend on the nature of the original tat mutation. Two of the mutants required cocultivation with tat-expressing cells to fully generate replication competent viruses, while a third mutant required only cocultivation with H9 cells. Sequencing of cDNA from cells infected with this last mutant demonstrated that the parental mutant sequence was retained and that genotypic revertants to the wild-type as well as new mutant sequences were generated. These results suggest that tat-defective HIV-1 provirus can be activated by UV and can subsequently revert to wild-type virus. This study raises the possibility that UV exposure of immune cells in the skin plays a role in the activation of defective HIV-1 in vivo.