In?vitro and in?vivo study on the effect of antifungal agents on hematopoietic cells in mice

Liposomal amphotericin B, voriconazole, and caspofungin are currently used for systemic and severe fungal infections. Patients with malignant diseases are treated with granulocyte-colony stimulating factor (G-CSF) for the recovery of granulocytes after chemotherapy or hematopoietic cell (HC) transplantation. Since they have a high incidence of fungal infections, they inevitably receive antifungal drugs for treatment and prophylaxis. Despite their proven less toxicity for various cell types comparatively with amphotericin B and the decrease in the number of leukocytes that has been reported as a possible complication in clinical studies, the effect of liposomal amphotericin B, voriconazole, and caspofungin on HCs has not been clarified. The present study aimed to examine the in vitro and in vivo effect of these three modern antifungals on HCs. Colony-forming unit (CFU) assays of murine bone marrow cells were performed in methylcellulose medium with or without cytokines and in the presence or absence of various concentrations of liposomal amphotericin B, voriconazole, and caspofungin. In the in vivo experiments, the absolute number of granulocytes was determined during leukocyte recovery in sublethally irradiated mice receiving each antifungal agent separately, with or without G-CSF. In vitro, all three antifungal drugs were nontoxic and, interestingly, they significantly increased the number of CFU-granulocyte-macrophage colonies in the presence of cytokines, at all concentrations tested. This was contrary to the concentration-dependent toxicity and the significant decrease caused by conventional amphotericin B. In vivo, the number of granulocytes was significantly higher with caspofungin plus G-CSF treatment, higher and to a lesser extent higher, but not statistically significantly, with voriconazole plus G-CSF and liposomal amphotericin B plus G-CSF treatments, respectively, as compared with G-CSF alone. These data indicate a potential synergistic effect of these antifungals with the cytokines, in vitro and in vivo, with subsequent positive effect on hematopoiesis.     

Mutation rates in humans. II. Sporadic mutation-specific rates and rate of detrimental human mutations inferred from hemophilia B

We estimated the rates per base per generation of specific types of mutations, using our direct estimate of the overall mutation rate for hemophilia B and information on the mutations present in the United Kingdom's population as well as those reported year by year in the hemophilia B world database. These rates are as follows: transitions at CpG sites 9.7x10-8, other transitions 7.3x10-9, transversions at CpG sites 5.4x10-9, other transversions 6.9x10-9, and small deletions/insertions causing frameshifts 3.2x10-10. By taking into account the ratio of male to female mutation rates, the above figures were converted into rates appropriate for autosomal DNA-namely, 1.3x10-7, 9.9x10-9, 7.3x10-9, 9.4x10-9, 6.5x10-10, where the latter is the rate for all small deletion/insertion events. Mutation rates were also independently estimated from the sequence divergence observed in randomly chosen sequences from the human and chimpanzee X and Y chromosomes. These estimates were highly compatible with those obtained from hemophilia B and showed higher mutation rates in the male, but they showed no evidence for a significant excess of transitions at CpG sites in the spectrum of Y-sequence divergence relative to that of X-chromosome divergence. Our data suggest an overall mutation rate of 2.14x10-8 per base per generation, or 128 mutations per human zygote. Since the effective target for hemophilia B mutations is only 1.05% of the factor IX gene, the rate of detrimental mutations, per human zygote, suggested by the hemophilia data is approximately 1.3.     

Peculiarities of the Na+/d-glucose cotransport system in necturus renal tubules

The effects of d-glucose addition to a glucose-free luminal perfusate were investigated in the proximal tubule of Necturus kidney, by electrophysiological techniques. The main findings are: (1) In the presence of sodium, d-glucose produces $\text{10.5}\text{mV}\text{± 1.1}$ (S.E.) depolarization. (2) Phlorizin reduces the magnitude of this response to $\text{2.1 ± 0.1}\text{mV}$. (3) The glucose-evoked depolarization, $\text{ΔV}{}_{\mathrm{<mtext>G</mtext>}}$, does not alter the intracellular K⁺ activity nor is it affected by peritubular addition of ouabain. (4) Isosmotic reduction of Na⁺ concentration in luminal perfusate from 95 to 2 mmol/l (choline or Li⁺ substituting for Na⁺) does not change the magnitude of $\text{ΔV}{}_{\mathrm{<mtext>G</mtext>}}$; complete removal of sodium from the lumen lowers the value of $\text{ΔV}{}_{\mathrm{<mtext>G</mtext>}}$ ($\text{3.2 ± 0.2}\text{mV}$) but the response is not abolished. This observation suggests that the d-glucose carrier of renal tubules in Necturus is poorly specific with regard to the cotransported cation species.     

It's a knockout!

'It's a Knockout!' provides an update of some of the latest mouse knockouts in TBASE (http://www.jax.org/tbase/ and Ref. 1). The column provides a concise phenotypic profile of novel mutants and renders their complete characterization directly accessible to Web users, via unique and unchanging accession numbers (TBASE identities). Where possible, interesting knockouts will be grouped according to gene families, application or phenotypic similarities.     

High plasma concentrations of acyl-coenzyme A binding protein (ACBP) predispose to cardiovascular disease: Evidence for a phylogenetically conserved proaging function of ACBP

Autophagy defects accelerate aging, while stimulation of autophagy decelerates aging. Acyl-coenzyme A binding protein (ACBP), which is encoded by a diazepam-binding inhibitor (DBI), acts as an extracellular feedback regulator of autophagy. As shown here, knockout of the gene coding for the yeast orthologue of ACBP/DBI (ACB1) improves chronological aging, and this effect is reversed by knockout of essential autophagy genes (ATG5, ATG7) but less so by knockout of an essential mitophagy gene (ATG32). In humans, ACBP/DBI levels independently correlate with body mass index (BMI) as well as with chronological age. In still-healthy individuals, we find that high ACBP/DBI levels correlate with future cardiovascular events (such as heart surgery, myocardial infarction, and stroke), an association that is independent of BMI and chronological age, suggesting that ACBP/DBI is indeed a biomarker of “biological” aging. Concurringly, ACBP/DBI plasma concentrations correlate with established cardiovascular risk factors (fasting glucose levels, systolic blood pressure, total free cholesterol, triglycerides), but are inversely correlated with atheroprotective high-density lipoprotein (HDL). In mice, neutralization of ACBP/DBI through a monoclonal antibody attenuates anthracycline-induced cardiotoxicity, which is a model of accelerated heart aging. In conclusion, plasma elevation of ACBP/DBI constitutes a novel biomarker of chronological aging and facets of biological aging with a prognostic value in cardiovascular disease.     

Retrograde coronary venous perfusion at low pressure

The effectiveness of localized retrograde coronary venous perfusion (RCVP) in preventing or reversing myocardial ischemia after acute ligation of a coronary artery is described. Ten domestic pigs (Group I) underwent aorto left anterior coronary vein grafting with RCVP at systemic pressure. In another set of ten pigs (Group II), the coronary vein was similarly grafted, but the proximal end of the graft was perfused with oxygenated blood by means of an external pump at reduced pressure and flow. In both groups, RCVP successfully reversed the mechanical or electrical effects produced by 2 to 5 minutes of acute arterial ligation. After several hours, Group I animals showed evidence of acute ischemia and developing infarction. Group II animals, however, were maintained for 7 hours or more with regional RCVP and no evidence of ischemia. Retrograde coronary venous perfusion at reduced pressure may be more effective than perfusion at systemic pressure in providing myocardial blood flow.     

Mechanisms of 1,25(OH)2D3-induced rapid changes of membrane potential in proximal tubule: Role of Ca2+-dependent K+ channels

Summary Eleven different secosteroids or steroids (10^–10 to 10^–8 m) were acutely and reversibly introduced in solutions delivered to the lumen of single proximal tubules of the amphibianNecturus kidney while recording basolateral cell membrane potentialV _m. Seven of these molecules (1,25(OH)₂D₃, 25(OH)D₃, 24,25(OH)₂D₃, 5,6-trans-25(OH)D₃, 19-diol-cholesterol, estradiol and testosterone) resulted in changes ofV _m (V _m) occurring in a few seconds, the largest V _m being observed with 1,25(OH)₂D₃, +6.5±0.75 mV (n=19); these seven (seco)steroids but not the four inactive sterols (vitamin D₃, cholesterol, 1D₃ and aldosterone) possess a hydroxyl group on at least one carbon of the C₁₇ to C₂₅ lateral chain of the sterol ring. The V _m effect was present in Na⁺-free or Cl^–-free media, but it was abolished in HCO₃-free media. Depolarization of cell membrane potential by addition of glucose, 11mm, in luminal perfusion fluid abolished the 1,25(OH)₂D₃-evoked V _m effect, suggesting dependence of the latter on the absolute value of membrane potential. Barium, a blocking agent of K⁺ conductances, suppressed the 1,25(OH)₂D₃-evoked V _m effect, even when the proper effects of barium of cell membrane potential were canceled by current clamp. Pretreatment with quinine, a putative blocker of Ca²⁺-dependent K⁺ channels also abolished the 1,25(OH)₂D₃-evoked depolarization. Such observations are consistent with the presence of Ca²⁺-dependent K⁺ channels at the apical cell membrane of the proximal tubule, these channels being inactivated by 1,25(OH)₂D₃ and probably by other (seco)steroids.     

Genome-independent effects of 1,25-dihydroxy vitamin D-3 on membrane potential

Cell membrane potential, $\text{V}{}_{\mathrm{<mtext>m</mtext>}}$, was monitored in rabbit hypertrophic cartilage metatarsals, amphibian proximal tubule and muscle cells during application of 1,25-dihydroxy vitamin D-3, 25-hydroxy vitamin D-3 or cholesterol (10^?10M). 1,25-Dihydroxy vitamin D-3 elicited quick variations of $\text{V}{}_{\mathrm{<mtext>m</mtext>}}$ (in less than 1 min) in proximal tubular cells (whether injected in the lumen or in peritubular capillaries) and in cartilage. The precursor 25-hydroxy vitamin D-3 and cholesterol produced a small shift of $\text{V}{}_{\mathrm{<mtext>m</mtext>}}$ in proximal tubule only when applied from the luminal side, but this change was significantly smaller than that observed with 1,25-dihydroxy vitamin D-3. Muscle cells were unresponsive to both metabolites and cholesterol. It is concluded that rapid effects of 1,25-dihydroxy vitamin D-3 on $\text{V}{}_{\mathrm{<mtext>m</mtext>}}$, in target cells, are specific, most likely due to permeability changes and not related to nuclear protein synthesis; they may contribute to early modulation of cell function.     

Studies on the Heat Resistance of Bacteria, with Particular Reference to the Genus Microbacterium: I. A New Technique using Solid Media

Summary: The new technique depends upon spreading bacterial cells over the surface of an agar medium in a Petri dish. The cells are heated, cooled, grown and counted in situ . The plates are preheated on the surface of a water bath and held for the required time on the surface of a second bath at the desired temperature. They are cooled by cold water jetted on to the base. The experimental control of factors affecting the accuracy of the method are discussed and applications suggested.