HIV regulatory proteins: potential targets for therapeutic intervention.

With the incidence of HIV infection on the rise worldwide, it is obvious that new approaches must be taken to halt the spread of disease. Unfortunately, this is no easy task; of all retroviruses studied to date HIV remains the most complex in terms of genomic organization, regulation of gene expression, and replication. However, as the mechanism of action of the unique viral regulatory proteins is deciphered, new windows of opportunity for attacking the virus like cycle are opened. The essential regulatory function served by both Tat and Rev transacting regulatory proteins makes them attractive targets for prophylactic and therapeutic intervention. This review will focus on our current understanding of Tat and Rev function.     

Growth factor receptors and related signalling pathways as targets for novel treatment strategies of hepatocellular cancer

Growth factors and their corresponding receptors are commonly overexpressed and/or dysregulated in many cancers including hepatocellular cancer （HCC）. Clinical trials indicate that growth factor receptors and their related signalling pathways play important roles in HCC cancer etiology and progression, thus providing rational targets for innovative cancer therapies. A number of strategies including monoclonal antibodies, tyrosine kinase inhibitors （＂small molecule inhibitors＂） and antisense oligonucleotides have already been evaluated for their potency to inhibit the activity and downstream signalling cascades of these receptors in HCC. First clinical trials have also shown that multi-kinase inhibition is an effective novel treatment strategy in HCC. In this respect sorafenib, an inhibitor of Raf-, VEGF- and PDGF-signalling, is the first multi-kinase inhibitor that has been approved by the FDA for the treatment of advanced HCC. Moreover, the serine-threonine kinase of mammalian target of rapamycin （mTOR） upon which the signalling of several growth factor receptors converge plays a central role in cancer cell proliferation, roTOR inhibition of HCC is currently also being studied in preclinical trials. As HCCs represent hypervascularized neoplasms, inhibition of tumour vessel formation via interfering with the VEGF/VEGFR system is another promising approach in HCC treatment. This review will summarize the current status of the various growth factor receptor-based treatment strategies and in view of the multitude of novel targeted approaches, the rationale for combination therapies for advanced HCC treatment will also be taken into account.     

Mechanisms of tubulointerstitial injury in the kidney: final common pathways to end-stage renal failure

There are many different glomerular disorders, including glomerulonephritis, diabetic nephropathy, and hypertensive nephrosclerosis. However, once glomerular damage reaches a certain threshold, the progression of renal disease is consistent and irreversible. Recent studies emphasized the crucial role of tubulointerstitial injury as a mediator of progression of kidney disease. One common mechanism that leads to renal failure via tubulointerstitial injury is massive proteinuria. Accumulating evidence suggests critical effects of filtered macromolecules on tubular cells, including lysosomal rupture, energy depletion, and tubular injury directly induced by specific components such as complement components. Another common mechanism is chronic hypoxia in the tubulointerstitium. Tubulointerstitial damage results in the loss of peritubular capillaries, impairing blood flow delivery. Interstitial fibrosis also impairs oxygen diffusion and supply to tubular cells. This induces chronic hypoxia in this compartment, rendering a vicious cycle. Development of novel therapeutic approaches against these final common pathways will enable us to target any types of renal disease.     

The lymphomas: molecular pathways and novel therapeutic targets

In recent years, several molecular mechanisms involved in promoting cancer cell survival and growth have been identified. These discoveries helped in designing and testing novel drugs that target specific cellular pathways. In this review, we focus on new molecular targets that are currently being explored for the treatment of non-Hodgkin's lymphoma and Hodgkin's lymphoma.     

Systemic hypertension among women with uterine leiomyomata: potential final common pathways of target end-organ remodeling

The uterus and the heart share a common structure and may remodel in a similar fashion, albeit temporally distinct. The authors investigated the prevalence of systemic hypertension in women with uterine leiomyomata (fibroids) and compared the prevalence in women undergoing hysterectomy for other reasons as well as in age-matched women from the National Health and Nutrition Examination Survey III (NHANES III). A total of 584 women, 205 with leiomyomata in 1999 (group A) and 379 who underwent hysterectomy for a variety of reasons in 2000 (group B) at Advocate Christ Medical Center were included. Presence of leiomyomata was confirmed by pathology. Hypertension was defined as blood pressure > or = 140/90 mm Hg or history of hypertension with or without medication use. The prevalence of hypertension in group A and B patients with leiomyomata compared with NHANES III overall was 48.6% vs. 24% (p<0.001), in African Americans 55.5% vs. 32.4% (p<0.001), and in Caucasians 51.1% vs. 23.3% (p<0.001). Leiomyomata were more frequent among hypertensive than normotensive women (57% vs. 27%). Caucasian and African-American women with leiomyomata were significantly younger and more likely to use hormone replacement therapy than others. Thus there appears to be an association between leiomyomata and hypertension, which needs to be explored in future prospective trials.     

肺泡上皮细胞间质转化及其信号转导途径在肺纤维化中的作用

目的以TGF-β1诱导A549细胞出现上皮细胞-间质细胞转化（epithelial-mesenchymal transition,EMT）,探讨EMT过程及其信号转导途径在肺纤维化中的作用。方法体外培养A549细胞,以TGF-β1进行干预,收集不同时段的细胞,用荧光实时定量PCR检测上皮及间质细胞标志物的mRNA表达,Western blot检测上皮及间质细胞的标志物以及信号转导蛋白P-Smad2/3、Snail1、Snail2的表达,应用倒置相差显微镜观察TGF-β1干预前后细胞形态学的变化。结果 TGF-β1干预后,A549细胞上皮细胞标志物的mRNA和蛋白表达下调（P〈0.05）,间质细胞标志物的mRNA和蛋白表达上调（P〈0.05）;信号转导蛋白P-Smad2/3、Snail1上调（P〈0.05）,snail2弱表达（P〉0.05）;倒置相差显微镜观察到TGF-β1干预后A549细胞由鹅卵石状变为梭形,形态如同肌纤维母细胞。结论 TGF-β1可以在体外诱导肺泡上皮细胞向间质细胞转化,其机制与Smad 2/3、Snail1信号转导途径相关,提示肺泡上皮细胞向间质细胞的转化是肺纤维化发生的直接原因,而Smad 2/3、Snail1信号转导途径很可能是关键环节。通过诱导或促进肺泡上皮细胞向间质细胞转化,可以促使肺纤维化的发病,而通过阻断这一过程及其下游的信号转导通路有望治疗肺纤维化。     

The anti-leukaemic activity of novel synthetic naphthoquinones against acute myeloid leukaemia: induction of cell death via the triggering of multiple signalling pathways

Naphthoquinones, such as menadione, display lower toxicity than anthracyclins used in cancer chemotherapy. Novel anti-leukaemic compounds comprised of chloro-amino-phenyl naphthoquinones with substitutions on the benzoic ring were developed. Structure–activity relationship studies indicated that the analogue with both methyl and amine substitutions (named TW-92) was the most efficient in killing leukaemic cells. Treatment of U-937 promonocytic cells with TW-92 induced apoptotic or necrotic cell death, dependent on incubation and dose conditions. TW-92 induced rapid phosphorylation of p38 mitogen-activated protein kinase (p38^MAPK) and of extracellular signal-regulated protein kinases (ERK1/2). The generation of apoptosis was preceded by intracellular H₂O₂ accumulation accompanied by glutathione depletion, the former inhibited by di-phenyl-iodonium (DPI), an inhibitor of NADPH oxidase. TW-92 induced swelling of isolated rat liver mitochondria, indicative of a direct effect on mitochondria. Apoptosis in intact cells was accompanied by a decrease in mitochondrial membrane potential, cytochrome c release and caspase activation. In addition, the level of Mcl-1, an anti-apoptotic regulatory protein, was down-regulated, whereas the expression of the pro-apoptotic BAX was elevated. Finally, TW-92 exerted strong pro-apoptotic and necrotic effects in primary acute myeloid leukaemia samples when given in submicromolar concentrations. Together, these findings demonstrate that TW-92 may provide an effective anti-leukaemic strategy.     

Duodenal alkaline secretion: its mechanisms and role in mucosal protection against gastric acid

Duodenal mucosa, especially its proximal portion, is exposed to intermittent pulses of gastric acid (H+). This review summarises the mechanisms of duodenal bicarbonate (HCO3-) secretion and their role in protecting duodenal epithelium against gastric H+. Duodenal epithelium is a leaky barrier against gastric H+, which diffuses into duodenocytes, but fails to damage them due to: (a) an enhanced expression of cyclooxygenase, producing protective prostaglandins and expression of nitric oxide synthase, releasing nitric oxide, both stimulating duodenal HCO3- secretion and (b) the release of several neurotransmitters also stimulating HCO3- secretion such as vasoactive intestinal peptide, pituitary adenylate cyclase-activating polypeptide, acetylcholine and melatonin. At the apical duodenocyte membrane, several HCO3-/Cl- anion exchangers operate in response to luminal H+ to extrude HCO3- into duodenal lumen. In baso-lateral duodenocyte membrane, both non-electrogenic and electrogenic Na+-HCO3- cotransporters are activated after exposure of duodenum to gastric H+, causing inward movement of HCO3- from extracellular fluid to duodenocytes. There are also at least three Na+/H+ exchangers, eliminating H+ which diffused into these cells. The Helicobacter pylori infection with gastric metaplasia in the duodenum and bacterium inoculation results in the inhibition of HCO3- secretion by its endogenous inhibitor dimethyl arginine, resulting in ulcerogenesis.     

Bidirectional role of tumor necrosis factor-alpha in coronary microembolization: progressive contractile dysfunction versus delayed protection against infarction

In patients with unstable angina, plaque rupture and coronary microembolization (ME) can precede complete coronary artery occlusion and impending infarction. ME-induced microinfarcts initiate an inflammatory reaction with increased tumor necrosis factor-alpha (TNF-alpha) expression, resulting in progressive contractile dysfunction. However, TNF-alpha is not only a negative inotrope but can also protect the myocardium against infarction. In anesthetized pigs, we studied whether ME protects against infarction when TNF-alpha expression is increased. ME (group1; n=7) was induced by intracoronary infusion of microspheres (42 microm; 3000 per mL/min inflow). Controls (group 2; n=8) received saline. Groups 3 and 4 (n=4 each) were pretreated with ovine TNF-alpha antibodies (25 mg/kg body weight) 30 minutes before ME or placebo, respectively. Ischemia (90 minutes) was induced 6 hours after ME when TNF-alpha was increased (66+/-21 pg/g wet weight; mean+/-SEM) or after placebo (TNF-alpha, 21+/-10 pg/g; P<0.05). Infarct size (percentage area at risk) was determined after 2 hours of reperfusion (triphenyl tetrazolium chloride staining). ME decreased systolic wall thickening progressively over 6 hours (group 1 versus group 2, 65+/-4% versus 90+/-1%; percentage of baseline; P<0.05). TNF-alpha antibodies attenuated the progressive decrease in systolic wall thickening following ME (group 3, 77+/-5% of baseline; P<0.05 versus group 1) with no effect in controls (group 4; 90+/-8% of baseline). With ME, infarct size was decreased to 18+/-4% versus 33+/-4% in group 2 (P<0.05). The infarct size reduction was abolished by TNF-alpha antibodies (group 3 versus group 4, 29+/-3% versus 35+/-5%). In ME, TNF-alpha is responsible for both progressive contractile dysfunction and delayed protection against infarction.     

The signalling pathway of CaMKII-mediated apoptosis and necrosis in the ischemia/reperfusion injury

Ca²⁺-calmodulin-dependent protein kinase II (CaMKII) plays an important role mediating apoptosis/necrosis during ischemia-reperfusion (IR). We explored the mechanisms of this deleterious effect. Langendorff perfused rat and transgenic mice hearts with CaMKII inhibition targeted to sarcoplasmic reticulum (SR-AIP) were subjected to global IR. The onset of reperfusion increased the phosphorylation of Thr¹⁷ site of phospholamban, without changes in total protein, consistent with an increase in CaMKII activity. Instead, there was a proportional decrease in the phosphorylation of Ser2815 site of ryanodine receptors (RyR2) and the amount of RyR2 at the onset of reperfusion, i.e. the ratio Ser2815/RyR2 did not change. Inhibition of the reverse Na⁺/Ca²⁺exchanger (NCX) mode (KBR7943) diminished phospholamban phosphorylation, reduced apoptosis/necrosis and enhanced mechanical recovery. CaMKII-inhibition (KN-93), significantly decreased phospholamban phosphorylation, infarct area, lactate dehydrogenase release (LDH) (necrosis), TUNEL positive nuclei, caspase-3 activity, Bax/Bcl-2 ratio and Ca²⁺-induced mitochondrial swelling (apoptosis), and increased contractile recovery when compared with non-treated IR hearts or IR hearts pretreated with the inactive analog, KN-92. Blocking SR Ca²⁺ loading and release (thapsigargin/dantrolene), mitochondrial Ca²⁺ uniporter (ruthenium red/RU360), or mitochondrial permeability transition pore (cyclosporine A), significantly decreased infarct size, LDH release and apoptosis. SR-AIP hearts failed to show an increase in the phosphorylation of Thr¹⁷ of phospholamban at the onset of reflow and exhibited a significant decrease in infarct size, apoptosis and necrosis respect to controls. The results reveal an apoptotic-necrotic pathway mediated by CaMKII-dependent phosphorylations at the SR, which involves the reverse NCX mode and the mitochondria as trigger and end effectors, respectively, of the cascade.     

Clinical review 125: The insulin receptor and its cellular targets

The pleiotropic actions of insulin are mediated by a single receptor tyrosine kinase. Structure/function relationships of the insulin receptor have been conclusively established, and the early steps of insulin signaling are known in some detail. A generally accepted paradigm is that insulin receptors, acting through insulin receptor substrates, stimulate the lipid kinase activity of phosphatidylinositol 3-kinase. The rapid rise in Tris-phosphorylated inositol (PIP(3)) that ensues triggers a cascade of PIP(3)-dependent serine/threonine kinases. Among the latter, Akt (a product of the akt protooncogene) and atypical protein kinase C isoforms are thought to be involved in insulin regulation of glucose transport and oxidation; glycogen, lipid, and protein synthesis; and modulation of gene expression. The presence of multiple insulin-regulated, PIP(3)-dependent kinases is consistent with the possibility that different pathways are required to regulate different biological actions of insulin. Additional work remains to be performed to understand the distal components of insulin signaling. Moreover, there exists substantial evidence for insulin receptor substrate- and/or phosphatidylinositol 3-kinase-independent pathways of insulin action. The ultimate goal of these investigations is to provide clues to the pathogenesis and treatment of the insulin resistant state that is characteristic of type 2 diabetes.