mCCDcl1 cells show plasticity consistent with the ability to transition between principal and intercalated cells.

The cortical collecting duct of the mammalian kidney plays a critical role in the regulation of body volume, sodium pH, and osmolarity and is composed of two distinct cells types, principal cells and intercalated cells. Each cell type is detectable in the kidney by the localization of specific transport proteins such as aquaporin 2 (Aqp2) and epithelial sodium channel (ENaC) in principal cells and V-ATPase B1 and connexin 30 (Cx30) in intercalated cells. mCCDcl1 cells have been widely used as a mouse principal cell line on the basis of their physiological characteristics. In this study, the mCCDcl1 parental cell line and three sublines cloned from isolated single cells (Ed1, Ed2, and Ed3) were grown on filters to assess their transepithelial resistance, transepithelial voltage, equivalent short circuit current and expression of the cell-specific markers Aqp2, ENaC, V-ATPaseB1, and Cx30. The parental mCCDcl1 cell line presented amiloride-sensitive electrogenic sodium transport indicative of principal cell function; however, immunocytochemistry and RT-PCR showed that some cells expressed the intercalated cell-specific markers V-ATPase B1 and Cx30, including a subset of cells also positive for Aqp2 and ENaC. The three subclonal lines contained cells that were positive for both intercalated and principal cell-specific markers. The vertical transmission of both principal and intercalated cell characteristics via single cell cloning reveals the plasticity of mCCDcl1 cells and a direct lineage relationship between these two physiologically important cell types and is consistent with mCCDcl1 cells being precursor cells.

Physiological roles of glucocorticoids during early embryonic development of the zebrafish (Danio rerio).

While glucocorticoids (GCs) are known to be present in the zebrafish embryo, little is known about their physiological roles at this stage. We hypothesised that GCs play key roles in stress response, hatching and swim activity during early development. To test this, whole embryo cortisol (WEC) and corticosteroid-related genes were measured in embryos from 6 to 120 h post fertilisation (hpf) by enzyme linked immunosorbent assay (ELISA) and quantitative real-time polymerase chain reaction (qRT-PCR). Stress response was assessed by change in WEC following stirring, hypoxia or brief electrical impulses applied to the bathing water. The impact of pharmacological and molecular GC manipulation on the stress response, spontaneous hatching and swim activity at different stages of development was also assessed. WEC levels demonstrated a biphasic pattern during development with a decrease from 0 to 36 hpf followed by a progressive increase towards 120 hpf. This was accompanied by a significant and sustained increase in the expression of genes encoding cyp11b1 (GC biosynthesis), hsd11b2 (GC metabolism) and gr (GC receptor) from 48 to 120 hpf. Metyrapone (Met), an inhibitor of 11ß-hydroxylase (encoded by cyp11b1), and cyp11b1 morpholino (Mo) knockdown significantly reduced basal and stress-induced WEC levels at 72 and 120 hpf but not at 24 hpf. Spontaneous hatching and swim activity were significantly affected by manipulation of GC action from approximately 48 hpf onwards. We have identified a number of key roles of GCs in zebrafish embryos contributing to adaptive physiological responses under adverse conditions. The ability to alter GC action in the zebrafish embryo also highlights its potential value for GC research.

Renal pericytes: multifunctional cells of the kidneys.

Pericytes have become a hot topic in renal biology. They play a critical physiological role in vessel development, maintenance and remodelling through active communication with their vascular partners-endothelial cells-and modulation of extracellular matrix proteins. Multiple functions for renal pericytes have been described; specialised perivascular populations participate in glomerular filtration, regulate medullary blood flow and contribute to kidney fibrosis by differentiation into collagen-generating myofibroblasts. Interestingly, the origin of renin-producing cells of the juxtaglomerular region is attributed to the perivascular cell lineage; we have observed the coincidence of renin and pericyte marker expression during human kidney development. Finally, pericytes have been shown to share features with mesenchymal stem cells, which places them as potential renal progenitor cell candidates. Since renal diseases are often associated with microvascular complications, renal pericytes may emerge as new targets for the treatment of kidney disease.

Acyclovir and transmission of HIV-1 from persons infected with HIV-1 and HSV-2.

BACKGROUND: Most persons who are infected with human immunodeficiency virus type 1 (HIV-1) are also infected with herpes simplex virus type 2 (HSV-2), which is frequently reactivated and is associated with increased plasma and genital levels of HIV-1. Therapy to suppress HSV-2 reduces the frequency of reactivation of HSV-2 as well as HIV-1 levels, suggesting that suppression of HSV-2 may reduce the risk of transmission of HIV-1. METHODS: We conducted a randomized, placebo-controlled trial of suppressive therapy for HSV-2 (acyclovir at a dose of 400 mg orally twice daily) in couples in which only one of the partners was seropositive for HIV-1 (CD4 count, > or = 250 cells per cubic millimeter) and that partner was also infected with HSV-2 and was not taking antiretroviral therapy at the time of enrollment. The primary end point was transmission of HIV-1 to the partner who was not initially infected with HIV-1; linkage of transmissions was assessed by means of genetic sequencing of viruses. RESULTS: A total of 3408 couples were enrolled at 14 sites in Africa. Of the partners who were infected with HIV-1, 68% were women, and the baseline median CD4 count was 462 cells per cubic millimeter. Of 132 HIV-1 seroconversions that occurred after randomization (an incidence of 2.7 per 100 person-years), 84 were linked within couples by viral sequencing: 41 in the acyclovir group and 43 in the placebo group (hazard ratio with acyclovir, 0.92, 95% confidence interval [CI], 0.60 to 1.41; P=0.69). Suppression with acyclovir reduced the mean plasma concentration of HIV-1 by 0.25 log(10) copies per milliliter (95% CI, 0.22 to 0.29; P<0.001) and the occurrence of HSV-2-positive genital ulcers by 73% (risk ratio, 0.27; 95% CI, 0.20 to 0.36; P<0.001). A total of 92% of the partners infected with HIV-1 and 84% of the partners not infected with HIV-1 remained in the study for 24 months. The level of adherence to the dispensed study drug was 96%. No serious adverse events related to acyclovir were observed. CONCLUSIONS: Daily acyclovir therapy did not reduce the risk of transmission of HIV-1, despite a reduction in plasma HIV-1 RNA of 0.25 log(10) copies per milliliter and a 73% reduction in the occurrence of genital ulcers due to HSV-2. (ClinicalTrials.gov number, NCT00194519.)

Proteomic analysis of epidermal mucus from sea lice-infected Atlantic salmon, Salmo salar L.

Health diets that contain immunostimulants and other functional ingredients can strengthen the immune response in Atlantic salmon, Salmo salar, and thereby reduce sea lice, Lepeophtheirus salmonis, infection levels. Such diets can be used to supplement other treatments and will potentially reduce the need for delousing and medication. A sea lice infection trial was conducted on fish with an average weight of 215 g. One control diet and four experimental diets containing functional ingredients were produced. The diets were fed to salmon for 4 weeks before infection with sea lice copepodids. When lice had developed to chalimus III/IV, 88 fish per diet were examined for lice loads. Mucus samples from fish fed the different diets were taken before and after lice infection. Mass spectrometry-based proteomics was used to characterize the protein composition in the epidermal mucus of Atlantic salmon and to identify quantitative alterations in protein expression. Multivariate analysis of the generated data sets was performed to identify protein biomarkers. Putative biomarkers associated with functional feed intake and with sea lice infection have been identified and can form the basis for strategic validation experiments with selected functional feeds.

Identification of Grf1 on mouse chromosome 9 as an imprinted gene by RLGS-M.

Normal mammalian development requires a diploid combination of both haploid parental genomes. Uniparental disomy for certain segments of specific chromosomes results in aberrant development or prenatal lethality, indicating that the parental genomes have undergone modifications during gametogenesis. These modifications result in parent-of-origin specific expression for some genes, a phenomenon called genomic imprinting. Recent work with DNA methyltransferase deficient mice showed that differential methylation is the probable basis of the imprinted character of several genes. Screening for endogenous imprinted loci using restriction landmark genomic scanning with methylation sensitive enzymes (RLGS-M) identified eight imprinted RLGS (Irigs) candidate loci. Molecular analysis of the genomic region of one of the loci (Irigs2) resulted in the discovery of the paternally imprinted U2afbp-rs gene within a previously identified imprinted region on mouse chromosome 11 (refs 5, 7). This paper describes the characterisation of a novel imprinted RLGS-M locus, Irigs3, on mouse chromosome 9 (ref. 6). Within this locus we identified the Grf1 (also called Cdc25Mm) gene, which is homologous to the RAS-specific guanine nucleotide exchange factor gene, CDC25, in Saccharomyces cerevisiae. Grf1 is located about 30 kb downstream of the methylation imprinted site, identified by RLGS-M, and shows paternal allele specific expression in mouse brain, stomach and heart. Our results indicate that imprinting may have a role in regulating mitogenic signal transduction pathways during growth and development.

Isolated discrete upper septal thickening in a non-referral cat population of senior and young cats.

INTRODUCTION/OBJECTIVES: Discrete upper septal thickening (DUST) is a phenotype of elderly people. The cardiac phenotype in senior cats has been incompletely described. We aimed to characterize the echocardiographic phenotype of senior cats, specifically to determine prevalence of DUST and hypertrophic cardiomyopathy (HCM). ANIMALS: One hundred and forty-nine healthy, normotensive cats. MATERIALS AND METHODS: Prospective cross-sectional study. Senior (≥9 years) and young (<6 years) cats were recruited from non-referral population. We defined DUST as an isolated basilar septal bulge, and HCM as left ventricular wall thickness ≥6 mm. An interventricular septum ratio (basal-to-mid septal thickness ratio) was calculated. We assessed for associations between clinical and echocardiographic variables and DUST. Data are presented as mean (±SD), median (range), or frequency (percentage). RESULTS: One-hundred and two senior and 47 young cats were enrolled. Aortoseptal angle (AoSA) was steeper in senior cats (137° (±14.5) vs. 145° (±12.3) in young cats, P=0.002). Eighteen cats had DUST (18/149, 12%), fourteen senior, and four young cats (P=0.4). Cats with DUST had steeper AoSA (125° (±8.3) vs. 142° (±13.7), P<0.0001) and higher interventricular septum ratio (1.4 (1.2-2.0) vs. 1.0 (0.7-1.8)). Univariable analysis showed decreased odds of DUST with greater AoSA (OR 0.9, P<0.0001), age was not associated with DUST. Twenty-nine senior cats had HCM (28.4%). DISCUSSION/CONCLUSIONS: Prevalence of DUST was 12%. There was no association between age and DUST. Smaller/steeper AoSA was the main factor associated with DUST. There was a high prevalence of HCM in this senior population.

Prevalence of Trauma-Induced Hypocalcemia in the Prehospital Setting.

BACKGROUND: Recent data published by the Special Operations community suggest the Lethal Triad of Trauma should be changed to the Lethal Diamond, to include coagulopathy, acidosis, hypothermia, and hypocalcemia. The purpose of this study is to determine the prevalence of trauma-induced hypocalcemia in level I and II trauma patients. METHODS: This is a retrospective cohort study conducted at a level I trauma center and Special Operations Combat Medic (SOCM) training site. Adult patients were identified via trauma services registry from September 2021 to April 2022. Patients who received blood products prior to emergency department (ED) arrival were excluded from the study. Ionized calcium levels were utilized in this study. RESULTS: Of the 408 patients screened, 370 were included in the final analysis of this cohort. Hypocalcemia was noted in 189 (51%) patients, with severe hypocalcemia identified in two (<1%) patients. Thirty-two (11.2%) patients had elevated international normalized ratio (INR), 34 (23%) patients had pH <7.36, 21 (8%) patients had elevated lactic acid, and 9 (2.5%) patients had a temperature of <35°C. CONCLUSION: Hypocalcemia was prevalent in half of the trauma patients in this cohort. The administration of a calcium supplement empirically in trauma patients from the prehospital environment and prior to blood transfusion is not recommended until further data prove it beneficial.

Transgenic approaches to modification of cell and tissue function.

The past 18 months have seen rapid advances in the use of transgenic techniques for elucidating cellular mechanisms. The modification of gene, cellular and tissue function has been enhanced by developments in the use of antisense and ribozyme constructs, and by improvements in strategies for cell ablation and homologous recombination.

Relationship between pre-existing viral reservoirs and the re-emergence of plasma viremia after discontinuation of highly active anti-retroviral therapy.

We examined the pathogenic significance of the latent viral reservoir in the resting CD4+ T cell compartment of HIV-1-infected individuals as well as its involvement in the rebound of plasma viremia after discontinuation of highly active anti-retroviral therapy (HAART). Using heteroduplex mobility and tracking assays, we show that the detectable pool of latently infected, resting CD4+ T cells does not account entirely for the early rebounding plasma HIV in infected individuals in whom HAART has been discontinued. In the majority of patients examined, the rebounding plasma virus was genetically distinct from both the cell-associated HIV RNA and the replication-competent virus within the detectable pool of latently infected, resting CD4 + T cells. These results indicate the existence of other persistent HIV reservoirs that could prompt rapid emergence of plasma viremia after cessation of HAART and underscore the necessity to develop therapies directed toward such populations of infected cells.

[Generation of rat induced pluripotent stem cells: the analysis of reprogramming and culturing media].

The rat represents very important, superior in many respects to the mous, animal model for studying pharmacology, physiology, ageing, cardiovascular etc. However, numerous attempts to derive rat ES cells necessary to carry out loss-of-gene-function studies have not been successful thus far. Therefore rat induct pluripotent stem cells (or riPS) should provide a notable alternative to ES cell, allowing to study gene functions in this valuable animal model. Here we report an improved lentivirus-based riPS derivation protocol that makes use of small inhibitors of MEK and GSK3. We show that the excision of proviruses does not affect neither karyotype and pluripotency state of these cells. Also, we propose genetic tool for an improvement of the quality of riPS cells in culture. These data may prompt further iPS-based gene targeting in rat as well as the development iPS-based gene therapies, using this animal model.

Cellular immune responses and viral diversity in individuals treated during acute and early HIV-1 infection.

Immune responses induced during the early stages of chronic viral infections are thought to influence disease outcome. Using HIV as a model, we examined virus-specific cytotoxic T lymphocytes (CTLs), T helper cells, and viral genetic diversity in relation to duration of infection and subsequent response to antiviral therapy. Individuals with acute HIV-1 infection treated before seroconversion had weaker CTL responses directed at fewer epitopes than persons who were treated after seroconversion. However, treatment-induced control of viremia was associated with the development of strong T helper cell responses in both groups. After 1 yr of antiviral treatment initiated in acute or early infection, all epitope-specific CTL responses persisted despite undetectable viral loads. The breadth and magnitude of CTL responses remained significantly less in treated acute infection than in treated chronic infection, but viral diversity was also significantly less with immediate therapy. We conclude that early treatment of acute HIV infection leads to a more narrowly directed CTL response, stronger T helper cell responses, and a less diverse virus population. Given the need for T helper cells to maintain effective CTL responses and the ability of virus diversification to accommodate immune escape, we hypothesize that early therapy of primary infection may be beneficial despite induction of less robust CTL responses. These data also provide rationale for therapeutic immunization aimed at broadening CTL responses in treated primary HIV infection.

Isolation and initial characterization of the mammalian midbody.

Midbodies were isolated from synchronized cultures of Chinese hamster ovary (CHO) cells and their protein composition was studied by means of SDS PAGE. Gels of the midbodies included alpha and beta tubulins as major bands (approximately 30% of the total protein) and approximately 35 other bands, none of which constituted greater than 3.5% of the total protein. Extraction of the isolated midbodies with Sarkosyl NL-30- solubilized the midbody microtubules but left the central, dense matrix zone of the midbody intact. A protein doublet of approximately 115,000 mol wt was retained preferentially by the particulate fraction containing the matrix zones, indicating it to be a component of the matrix. The 115,000 mol wt doublet was also present in gels of isolated mitotic spindles from CHO cells. The overall protein composition of the isolated spindles was very similar to that of the isolated midbodies.

Terminal phase of cytokinesis in D-98s cells.

The events leading to the completion of cytokinesis after the formation of the midbody and intercellular bridge in D-98S cells were studied with light and electron microscopy. Pairs of daughter cells corresponding to different stages of cytokineses, as determined previously form time lapse films, were selected from embedded monolayers for serial sectioning. Separation of daughter cells is preceded by the reduction in diameter of the intercellular bridge from 1-1.5 mum to approx. 0.2 mum. Two processes contribute to this reduction: (a) The intercellular bridge becomes gradually thinner after telophase; a progressive breakdown of midbody structures accompanies this change; and (b) the more significant contribution to reduction in bridge diameter occurs through the localized constriction of a segment of the intercellular bridge.. The microtubules within the constricted portion of the bridge are forced closer together, and some microtubules disappear as this narrowing progresses. The plasma membrane over the narrowed segments is thrown into a series of wavelike ripples. Separation of daughter cells is achieved through movements of the cells which stretch and break the diameter-reduced bridge. The midbody is discarded after separation and begins to deteriorate. Occasional pairs of daughter cells were found in which incomplete karyokineses resulted in their nuclei being connected by a strand of nuclear material traversing the bridge and midbody. Such cells do not complete cytokinesis but merge together several hours after telophase. This merging of daughter cells coincides with the nearly complete breakdown of the midbody.

Role of enzymatic wall-softening in plant morphogenesis: hormonal induction in Achlya.

Sexual hormone A, which induces antheridial branching in male strains of Achlya, also elicits a rise in cellulase. The peak of induced cellulase corresponds in time with the appearance of branches that are the male sexual organ primordia; only those strains that branch in response to the hormone show a concomitant rise in cellulase. The response to the hormone is inhibited by compounds that block protein synthesis, for example, p-fluorophenylalanine and puronmycin. Vegetative branching, induced by substrates such as casein hydrolysate, is also accompanied by a rise in cellulase.

Hormonal control of sexual morphogenesis in achlya: dependence on protein and ribonucleic Acid syntheses.

The induction of the male sexual organ primordia (antheridial hyphae) by the steroid hormone antheridiol in the water mold Achlya ambisexualis requires both transcription and translation. Inhibition of either of these processes eliminates the expected increase in the production and release of the enzyme cellulase, which accompanies the formation of the antheridial hyphae.

Molecular cloning of a feline leukemia virus that induces fatal immunodeficiency disease in cats.

A replication-defective variant of feline leukemia virus was molecularly cloned directly from infected tissue and found to induce a rapid and fatal immunodeficiency syndrome in cats. Studies with cloned viruses also showed that subtle mutational changes would convert a minimally pathogenic virus into one that would induce an acute form of immunodeficiency. The data suggest that acutely pathogenic viruses may be selected against by current methods for isolation of the human and simian immunodeficiency viruses.

Mung bean nuclease cleaves Plasmodium genomic DNA at sites before and after genes.

Mung bean nuclease was found to cut the genomic DNA of the malaria parasite Plasmodium at positions before and after genes but not within gene-coding regions. This cleavage, which had nearly the preciseness of a restriction nuclease, required controlled conditions in the presence of formamide. Southern blot analysis showed that the coding areas for Plasmodium actin, circumsporozoite protein, histidine-rich protein, ribosomal RNA's, and tubulin are each cleaved from genomic DNA to yield a single major band on an agarose gel. DNA sequence data on several clones of mung bean nuclease cleavage products containing the gene for the circumsporozoite protein of Plasmodium falciparum confirmed that cleavage sites are before and after genes. Recognition and cleavage of DNA did not seem to be related to any primary sequence but may be related to structural features of the DNA duplex that demarcate genes. Mung bean nuclease-cleaved DNA could be inserted directly into a lambda expression vector, yielding a representative but small gene bank of intact gene fragments.

Genetic relationships determined by a DNA heteroduplex mobility assay: analysis of HIV-1 env genes.

The genetic diversity of human immunodeficiency virus (HIV) is a major concern thought to impact on immunologic escape and eventual vaccine efficacy. Here, simple and rapid methods are described for the detection and estimation of genetic divergence between HIV strains on the basis of the observation that DNA heteroduplexes formed between related sequences have a reduced mobility in polyacrylamide gels proportional to their degree of divergence. Reliable phylogenetic subtypes were assigned for HIV-1 strains from around the world. Relationships between viruses were closest when derived from the same or epidemiologically linked individuals. When derived from epidemiologically unlinked individuals, the relationships between viruses in a given geographic region correlated with the length of time HIV-1 had been detected in the population and the number of strains initiating widespread infection. Heteroduplex mobility analysis thus provides a tool to expedite epidemiological investigations by assisting in the classification of HIV and is readily applicable to the screening and characterization of other infectious agents and cellular genes.

A transgenic model of visceral obesity and the metabolic syndrome.

The adverse metabolic consequences of obesity are best predicted by the quantity of visceral fat. Excess glucocorticoids produce visceral obesity and diabetes, but circulating glucocorticoid levels are normal in typical obesity. Glucocorticoids can be produced locally from inactive 11-keto forms through the enzyme 11beta hydroxysteroid dehydrogenase type 1 (11beta HSD-1). We created transgenic mice overexpressing 11beta HSD-1 selectively in adipose tissue to an extent similar to that found in adipose tissue from obese humans. These mice had increased adipose levels of corticosterone and developed visceral obesity that was exaggerated by a high-fat diet. The mice also exhibited pronounced insulin-resistant diabetes, hyperlipidemia, and, surprisingly, hyperphagia despite hyperleptinemia. Increased adipocyte 11beta HSD-1 activity may be a common molecular etiology for visceral obesity and the metabolic syndrome.