γ-H2AX and other histone post-translational modifications in the clinic

Chromatin is a dynamic complex of DNA and proteins that regulates the flow of information from genome to end product. The efficient recognition and faithful repair of DNA damage, particularly double-strand damage, is essential for genomic stability and cellular homeostasis. Imperfect repair of DNA double-strand breaks (DSBs) can lead to oncogenesis. The efficient repair of DSBs relies in part on the rapid formation of foci of phosphorylated histone H2AX (γ-H2AX) at each break site, and the subsequent recruitment of repair factors. These foci can be visualized with appropriate antibodies, enabling low levels of DSB damage to be measured in samples obtained from patients. Such measurements are proving useful to optimize treatments involving ionizing radiation, to assay in vivo the efficiency of various drugs to induce DNA damage, and to help diagnose patients with a variety of syndromes involving elevated levels of γ-H2AX. We will survey the state of the art of utilizing γ-H2AX in clinical settings. We will also discuss possibilities with other histone post-translational modifications. The ability to measure in vivo the responses of individual patients to particular drugs and/or radiation may help optimize treatments and improve patient care. This article is part of a Special Issue entitled: Chromatin in time and space.     

Fast Calcium-Dependent Inactivation of Calcium Release-Activated Calcium Current (CRAC) in RBL-1 Cells

Fast inactivation of the Ca²⁺ release-activated Ca²⁺ current (I _CRAC) was studied using whole cell patch-clamp recordings in rat basophilic leukemia (RBL-1) cells. Application of hyperpolarizing voltage steps from the holding potential of 0 mV revealed that I _CRAC declined in amplitude over tens of milliseconds during steps more negative than −40 mV. This fast inactivation was predominantly Ca²⁺-dependent because first, it could be more effectively suppressed when BAPTA was included in the recording pipette instead of EGTA and second, replacing external Ca²⁺ with Sr²⁺ resulted in less inactivation. Recovery from inactivation was faster in the presence of BAPTA than EGTA. The extent of fast inactivation was independent of the whole cell I _CRAC amplitude, compatible with the notion that the inactivation arose from a local feedback inhibition by permeating Ca²⁺ ions only on the channel it permeated. Ca²⁺ release from stores did not affect fast inactivation, nor did FCɛRI receptor stimulation. Current clamp recordings showed that the majority of RBL cells had a membrane potential close to −90 mV following stimulation of FCɛRI receptors. Hence fast inactivation is likely to impact on the extent of Ca²⁺ influx through CRAC channels under physiological conditions and appears to be an important negative feedback process that limits Ca²⁺ increases. Received: 28 August 1998/Revised: 30 November 1998     

Comparing lipid remodeling of brown adipose tissue,white adipose tissue,and liver after one-week high fat diet intervention with quantitative Raman microscopy

Brown adipose tissue (BAT) consists of highly metabolically active adipocytes that catabolize nutrients to produce heat. Playing an active role in triacylglycerol (TAG) clearance, research has shown that dietary fatty acids can modulate the TAG chemistry deposition in BAT after weeks-long dietary intervention, similar to what has been shown in white adipose tissue (WAT). Our objective was to compare the influence of sustained, nonchronic dietary intervention (a 1-week interval) on WAT and interscapular BAT lipid metabolism and deposition in situ. We use quantitative, label-free chemical microscopy to show that 1 week of high fat diet (HFD) intervention results in dramatically larger lipid droplet (LD) growth in BAT (and liver) compared to LD growth in inguinal WAT (IWAT). Moreover, BAT showed lipid remodeling as increased unsaturated TAGs in LDs, resembling the dietary lipid composition, while WAT (and liver) did not show lipid remodeling on this time scale. Concurrently, expression of genes involved in lipid metabolism, particularly desaturases, was reduced in BAT and liver from HFD-fed mice after 1 week. Our data show that BAT lipid chemistry remodels exceptionally fast to dietary lipid intervention compared WAT, which further points towards a role in TAG clearance.     

Fed-batch fermentation of glucose to acetate by an improved strain ofClostridium thermoaceticum

The fed-batch approach to the production of acetate from glucose by an improved strain ofClostridium thermoaceticum resulted in better performance than the batch fermentation, especially in media containing an excess (3X) of nutrients and trace salts. At pH 6.6, 46 g/l acetic acid was produced in 192 hours with 93% substrate utilization. In contrast, batch fermentation under similar conditions resulted in a maximum of 35 g/l acetic acid with less than 82% substrate utilization.     

The Association of Human Apolipoprotein C-III Sialylation Proteoforms with Plasma Triglycerides

Introduction

Apolipoprotein C-III (apoC-III) regulates triglyceride (TG) metabolism. In plasma, apoC-III exists in non-sialylated (apoC-III_0a without glycosylation and apoC-III_0b with glycosylation), monosialylated (apoC-III₁) or disialylated (apoC-III₂) proteoforms. Our aim was to clarify the relationship between apoC-III sialylation proteoforms with fasting plasma TG concentrations.

Methods

In 204 non-diabetic adolescent participants, the relative abundance of apoC-III plasma proteoforms was measured using mass spectrometric immunoassay.

Results

Compared with the healthy weight subgroup (n = 16), the ratios of apoC-III_0a, apoC-III_0b, and apoC-III₁ to apoC-III₂ were significantly greater in overweight (n = 33) and obese participants (n = 155). These ratios were positively correlated with BMI z-scores and negatively correlated with measures of insulin sensitivity (S_i). The relationship of apoC-III₁ / apoC-III₂ with S_i persisted after adjusting for BMI (p = 0.02). Fasting TG was correlated with the ratio of apoC-III_0a / apoC-III₂ (r = 0.47, p<0.001), apoC-III_0b / apoC-III₂ (r = 0.41, p<0.001), apoC-III₁ / apoC-III₂ (r = 0.43, p<0.001). By examining apoC-III concentrations, the association of apoC-III proteoforms with TG was driven by apoC-III_0a (r = 0.57, p<0.001), apoC-III_0b (r = 0.56. p<0.001) and apoC-III₁ (r = 0.67, p<0.001), but not apoC-III₂ (r = 0.006, p = 0.9) concentrations, indicating that apoC-III relationship with plasma TG differed in apoC-III₂ compared with the other proteoforms.

Conclusion

We conclude that apoC-III_0a, apoC-III_0b, and apoC-III₁, but not apoC- III₂ appear to be under metabolic control and associate with fasting plasma TG. Measurement of apoC-III proteoforms can offer insights into the biology of TG metabolism in obesity.