Hongchang Gao

Application of 1H NMR‐based metabonomics in the study of metabolic profiling of human hepatocellular carcinoma and liver cirrhosis

1H nuclear magnetic resonance (NMR)‐based metabonomics was used to characterize metabolic profiles of liver cirrhosis (LC) and hepatocellular carcinoma (HCC). We found compared to healthy humans, LC and HCC sera had higher levels of acetate, N‐acetylglycoproteins, pyruvate, glutamine, α‐ketoglutarate, glycerol, tyrosine, 1‐methylhistidine and phenylalanine, together with lower levels of low‐density lipoprotein, isoleucine, valine, acetoacetate, creatine, choline and unsaturated lipids. Scores plot of pattern recognition analysis were capable of distinguishing LC and HCC patients from healthy humans. These results indicate that serum NMR spectra combined with pattern recognition analysis techniques offer an efficient, convenient way of depicting tumor biochemistry, which may be of great benefit to early diagnosis of human malignant diseases using single blood samples. (Cancer Sci 2009; 100: 782–785)

Metabonomic profiling of renal cell carcinoma: high-resolution proton nuclear magnetic resonance spectroscopy of human serum with multivariate data analysis.

Metabonomic profiling using proton nuclear magnetic resonance ((1)H NMR) spectroscopy and multivariate data analysis of human serum samples was used to characterize metabolic profiles in renal cell carcinoma (RCC). We found distinct, easily detectable differences between (a) RCC patients and healthy humans, (b) RCC patients with metastases and without metastases, and (c) RCC patients before and after nephrectomy. Compared to healthy human serum, RCC serum had higher levels of lipid (mainly very low-density lipoproteins), isoleucine, leucine, lactate, alanine, N-acetylglycoproteins, pyruvate, glycerol, and unsaturated lipid, together with lower levels of acetoacetate, glutamine, phosphatidylcholine/choline, trimethylamine-N-oxide, and glucose. This pattern was somewhat reversed after nephrectomy. Altered metabolite concentrations are most likely the result of the cells switching to glycolysis to maintain energy homeostasis following the loss of ATP caused by impaired TCA cycle in RCC. Serum NMR spectra combined with principal component analysis techniques offer an efficient, convenient way of depicting tumour biochemistry and stratifying tumours under different pathophysiological conditions. It may be able to assist early diagnosis and postoperative surveillance of human malignant diseases using single blood samples.

Systemic Metabolic Changes of Traumatic Critically III Patients Revealed by an NMR-Based Metabonomic Approach

Progression of critically ill patients from Systemic Inflammatory Response Syndrome (SIRS) to Multiple Organ Dysfunction Syndrome (MODS) accounts for more than 75% of deaths in adult surgical intensive care units. Currently, there is no practical clinical technique to predict the progression of SIRS or MODS. In this report, we describe an NMR-based metabonomic method to aid detection of these conditions based on abnormal metabolic signatures. We applied pattern recognition methods to analyze one-dimensional (1)H NMR spectra of SIRS and MODS patient sera. By using Principal Component Analysis (PCA) and Partial Least Squares-Discriminant Analysis (PLS-DA), we could distinguish critically ill patients (n = 52) from healthy controls (n = 26). After noise reduction by Orthogonal Signal Correction (OSC), PLS-DA was also able to clearly discriminate SIRS and MODS patients. The corresponding coefficients indicated that spectra responsible for the discrimination were located in delta3.06-3.86 NMR integral regions from SIRS, mainly composed of sugars, amino acids and glutamine signals, and delta1.18-1.3 and delta4.02-4.1 integral regions of MODS serum samples, principally consisted of various proton signals of fatty acyl chains and glycerol backbone of lipids, along with creatinine and lactate. Our results are consistent with the clinical observations that carbohydrate and amino acid levels changes in the early course of critical illness (SIRS stage) and significant disturbances in fat metabolism and development of organ abnormalities become the characteristics in the late stage (MODS). These data suggest that NMR-based metabonomic approach can be developed to diagnose the disease progress of critically ill patients.

A Metabonomic Comparison of Urinary Changes in Zucker and GK Rats

To further investigate pathogenesis and pathogenic process of type 2 diabetes mellitus (T2DM), we compared the urinary metabolic profiling of Zucker obese and Goto-kakizaki (GK) rats by NMR-based metabonomics. Principal component analysis (PCA) on urine samples of both models rats indicates markedly elevated levels of creatine/creatinine, dimethylamine, and acetoacetate, with concomitantly declined levels of citrate, 2-ketoglurarate, lactate, hippurate, and succinate compared with control rats, respectively. Simultaneously, compared with Zucker obese rats, the GK rats show decreased levels of trimethylamine, acetate, and choline, as well as increased levels of creatine/creatinine, acetoacetate, alanine, citrate, 2-ketoglutarate, succinate, lactate, and hippurate. This study demonstrates metabolic similarities between the two stages of T2DM, including reduced tricarboxylic acid (TCA) cycle and increased ketone bodies production. In addition, compared with Zucker obese rats, the GK rats have enhanced concentration of energy metabolites, which indicates energy metabolic changes produced in hyperglycemia stage more than in insulin resistance stage.

1H NMR-based metabonomic analysis of metabolic profiling in diabetic nephropathy rats induced by streptozotocin

Elucidation of the metabolic profiling in diabetic nephropathy (DN) rats is of great assistance for understanding the pathogenesis of DN. In this study, ¹H-nuclear magnetic resonance (NMR)-based metabonomics combined with HPLC measurements was used to quantitatively analyze the metabolic changes in urine and kidney extracts from diabetic 2-wk and 8-wk rats induced by streptozotocin (STZ). Pattern recognition analysis of either urine or kidney extracts indicated that the two diabetic groups were separated obviously from the control group, suggesting that the metabolic profiles of the diabetic groups were markedly different from the control. The diabetic 8-wk rats showed lower levels of creatine, dimethylamine, and higher levels of ascorbate, succinate, lactate, citrate, allantoin, 2-ketoglutarate, and 3-hydrobutyrate (3-HB) in the urine samples. Moreover, the diabetic 8-wk rats displayed lower levels of succinate, creatine, myo-inositol, alanine, lactate, and ATP, and higher levels of 3-HB and glucose in the kidney extracts. The observed metabolic changes imply the enhanced pathways of either lipid or ketone body synthesis and decreased pathways of either tricarboxylic acid cycle or glycolysis in DN rats compared with the control. Our results suggest that the energy metabolic changes are associated with the pathogenic process of DN.

Metabonomic analysis of the therapeutic effect of Zhibai Dihuang Pill in treatment of streptozotocin-induced diabetic nephropathy

ETHNOPHARMACOLOGICAL RELEVANCEnZhibai Dihuang Pill (ZDP) is one of ancient traditional Chinese medicines (TCMs), which is usually used for the treatment of kidney deficiency for thousands of years in China.nnnAIM OF THE STUDYnTraditional Chinese medicines (TCMs) usually operate in vivo through multi-components, multi-ways and multi-targets. However, the molecular mechanisms of TCMs remain unclear. In the present work, nuclear magnetic resonance (NMR)-based metabonomic analysis was used to evaluate the therapeutic effect of Zhibai Dihuang Pill (ZDP) on diabetic nephropathy (DN) rats induced by streptozotocin and to address the underlying molecular mechanism.nnnMATERIALS AND METHODSnMale rats were divided into three groups: control, DN and ZDP-treated DN (ZDP-DN), respectively. Based on (1)H NMR spectra of sera, urine and kidney extracts from the rats, principle component analysis (PCA) was performed to identify different metabolic profiles. Kidney portions and serum and urine samples were also subjected to histopathological or biochemical examination.nnnRESULTSnPCA scores plots demonstrate that the cluster of DN rats is separated from that of control rats, while some of ZDP-DN rats are located close to control rats, indicating that metabolic profiles of these ZDP-DN rats are restored toward those of control rats. Our results illustrate that ZDP treatment could lower the levels of lipids and 3-hydrobutyrate, and raise the level of lactate in sera of DN rats. Moreover, ZDP treatment could also reduce the levels of glucose, 3-hydrobutyrate and lactate, enhance the level of betaine in kidney tissues.nnnCONCLUSIONnOur study indicates that ZDP treatment can ameliorate DN symptoms by intervening in some dominating metabolic pathways, such as inhibiting glucose and lipid metabolism, enhancing methylamine metabolism. Our work may be of benefit to both evaluation of the therapeutic effect of TCM and elucidation of the underlying molecular mechanism.

Differential ubiquitin binding of the UBA domains from human c-Cbl and Cbl-b: NMR structural and biochemical insights

The Cbl proteins, RING‐type E3 ubiquitin ligases, are responsible for ubiquitinating the activated tyrosine kinases and targeting them for degradation. Both c‐Cbl and Cbl‐b have a UBA (ubiquitin‐associated) domain at their C‐terminal ends, and these two UBA domains share a high sequence similarity (75%). However, only the UBA from Cbl‐b, but not from c‐Cbl, can bind ubiquitin (Ub). To understand the mechanism by which the UBA domains specifically interact with Ub with different affinities, we determined the solution NMR structures of these two UBA domains, cUBA from human c‐Cbl and UBAb from Cbl‐b. Their structures show that these two UBA domains share the same fold, a compact three‐helix bundle, highly resembling the typical UBA fold. Chemical shift perturbation experiments reveal that the helix‐1 and loop‐1 of UBAb form a predominately hydrophobic surface for Ub binding. By comparing the Ub‐interacting surface on UBAb and its counterpart on cUBA, we find that the hydrophobic patch on cUBA is interrupted by a negatively charged residue Glu12. Fluorescence titration data show that the Ala12Glu mutant of UBAb completely loses the ability to bind Ub, whereas the mutation disrupting the dimerization has no significant effect on Ub binding. This study provides structural and biochemical insights into the Ub binding specificities of the Cbl UBA domains, in which the hydrophobic surface distribution on the first helix plays crucial roles in their differential affinities for Ub binding. That is, the amino acid residue diversity in the helix‐1 region, but not the dimerization, determines the abilities of various UBA domains binding with Ub.

Recombinant expression and characterization of an epididymis-specific antimicrobial peptide BIN1b/SPAG11E.

BIN1b was reported as an epididymis-specific beta-defensin antimicrobial peptide. In this paper, the recombinant BIN1b was expressed and purified by fusing with GB1-His tag. The size-exclusion gel filtration experiment indicated that the fusion protein GB1-BIN1b formed multimers at pH 7.4, and existed as monomer at pH 4.5. The oligomerization of GB1-BIN1b was only related to pH value, neither to NaCl concentration nor protein concentration. Far-UV circular dichroism (CD) spectra also showed the fusion protein had more ordered secondary structures at pH 4.5 than at pH 7.4, as a negative peak appeared around 218 nm indicative of typical beta-sheet. The 2D (15)N-(1)H heteronuclear single-quantum coherence (HSQC) spectra suggested that the fusion protein adopted a compact three-dimensional structure at pH 4.5. Colony forming unit (CFU) inhibition assay demonstrated that 25 microM fusion protein at pH 7.4 had an antimicrobial activity of 40% against E. coli K(12)D(31), which might imply the fusion protein functions as multimeric states. In conclusion, the GB1 fusion partner helps BIN1b form a stable homogenous conformation to facilitate subsequent structural determination without a significant effect on the antimicrobial activity.

Structure of a novel PTH‐related peptide hPTH′ and its interaction with the PTH receptor

We have previously shown that a recombinant human PTH fragment, Pro‐Pro‐[Arg11] hPTH (1–34)‐Pro‐Pro‐Asp (hPTH′), could be a potentially better and more cost‐effective therapeutic agent than PTH (1–34) on osteoporosis. In this report, we characterized the solution conformations of hPTH′ by NMR spectroscopy and modeled the interactions between the hPTH′ and the PTH receptor. By comparing it with PTH (1–34) structures and their respective interactions with the PTH receptor, we identified two segments of helix extending from Ile5 to Met8 and from Glu22 to Gln29 with a divided kink between the two helixes around Arg20. Mutated arginine makes hPTH′ fill the receptor cavity better as well as forms hydrogen bonds with Val193. Understanding the ligand receptor interactions will help us design small molecules to better mimic the activities of PTH. Copyright