Overexpression of hypoxia-inducible factor and metabolic pathways: possible targets of cancer

Cancer, the main cause of human deaths in the modern world is a group of diseases. Anticancer drug discovery is a challenge for scientists because of involvement of multiple survival pathways of cancer cells. An extensive study on the regulation of each step of these pathways may help find a potential cancer target. Up-regulated HIF-1 expression and altered metabolic pathways are two classical characteristics of cancer. Oxygen-dependent (through pVHL, PHDs, calcium-mediated) and independent (through growth factor signaling pathway, mdm2 pathway, HSP90) regulation of HIF-1α leads to angiogenesis, metastasis, and cell survival. The two subunits of HIF-1 regulates in the same fashion through different mechanisms. HIF-1α translation upregulates via mammalian target of rapamycin and mitogen-activated protein kinase signaling pathways, whereas HIF-1β through calmodulin kinase. Further, the stabilized interactions of these two subunits are important for proper functioning. Also, metabolic pathways crucial for the formation of building blocks (pentose phosphate pathway) and energy generation (glycolysis, TCA cycle and catabolism of glutamine) are altered in cancer cells to protect them from oxidative stress and to meet the reduced oxygen and nutrient supply. Up-regulated anaerobic metabolism occurs through enhanced expression of hexokinase, phosphofructokinase, triosephosphate isomerase, glucose 6-phosphate dehydrogenase and down-regulation of aerobic metabolism via pyruvate dehydrogenase kinase and lactate dehydrogenase which compensate energy requirements along with high glucose intake. Controlled expression of these two pathways through their common intermediate may serve as potent cancer target in future.     

Lowering of intraocular pressure by topical application of Daucus carota seed extract in rabbits

In normotensive rabbits topical application of Daucus carota seed extract at the concentration of 0.3, 0.6 and 1.2% resulted in mean IOP reduction of 19.33. 23.20 and 25.61% respectively from baseline. As no significant difference was observed between the change in IOP in 0.6 and 1.2% extract treated groups, 0.6% concentration was chosen for further evaluation in rabbits with experimentally elevated IOP. In water loaded rabbits, maximum mean IOP reduction with 0.6% extract was 29.39%, which was comparable to pilocarpine. In steroid pretreated rabbits, maximum mean IOP reduction was 30.27% from baseline, which was significantly higher than pilocarpine. The extract showed a comparatively slower onset of action however, the duration of action was comparable to pilocarpine in all the experimental models.     

Bilateral cleft lip and nasal repair

SUMMARY: The bilateral cleft lip and nasal repair has remained a challenging endeavor. Techniques have evolved to address concerns over unsatisfactory features and stigmata of the surgery. The authors present an approach to this complex clinical problem that modifies traditional repairs described by Millard and Manchester. The senior author (H.S.B.) has developed this technique with over 25 years of surgical experience dealing with the bilateral cleft lip. This staged lip and nasal repair provides excellent nasal projection, lip function, and aesthetic outcomes. Lip repair is performed at 3 months of age. Columellar lengthening is performed at approximately 18 months of age. A key component of this repair focuses on reconstruction of the central tubercle. A triangular prolabial dry vermilion flap is augmented by lateral lip vermilion flaps that include the profundus muscle of the orbicularis oris. This minimizes lateral lip segment sacrifice and provides improved central vermilion fullness, which is often deficient in traditional repairs. The authors present the surgical technique and examples of their clinical results.     

Evaluation of glucose sensitive affinity binding assay entrapped in fluorescent dissolved‐core alginate microspheres

The feasibility of dissolved‐core alginate‐templated fluorescent microspheres as “smart tattoo” glucose biosensors was investigated in simulated interstitial fluid (SIF). The sensor works on the principle of competitive binding and fluorescence resonance energy transfer. The sensor consists of multilayer thin film coated alginate microspheres incorporating dye‐labeled glucose receptor and competing ligand within the partially dissolved alginate core. In this study, different approaches for the sensing and detection chemistry were studied, and the response of encapsulated reagents was compared with the solution‐phase counterparts. The glucose sensitivity of the encapsulated TRITC‐Con A/FITC‐dextran (500 kDa) assay in DI water was estimated to be 0.26%/mM glucose while that in SIF was observed to be 0.3%/mM glucose. The glucose sensitivity of TRITC‐apo‐GOx/FITC‐dextran (500 kDa) assay was estimated to be 0.33%/mM glucose in DI water and 0.5%/mM glucose in SIF and both demonstrated a response in the range of 0–50 mM glucose. Therefore, it is hypothesized that the calcium ion concentration outside the microsphere (in the SIF) does not interfere with the response sensitivity. The sensor response was observed to exhibit a maximum response time of 120 s. The system further exhibited a sensitivity of 0.94%/mM glucose with a response in range of 0–50 mM glucose, using near‐infrared dyes (Alexa Fluor‐647‐labeled dextran as donor and QSY‐21‐conjugated apo‐GOx as acceptor), thereby making the sensor more amenable to in vivo use, when implanted in scattering tissue. Biotechnol. Bioeng. 2009; 104: 1075–1085. © 2009 Wiley Periodicals, Inc.     

Pharmacological inhibition of inducible nitric oxide synthase attenuates the development of seizures in mice

The present study has been designed to pharmacologically expound the significance of inducible nitric oxide synthase in the pathophysiological progression of seizures using mouse models of chemically induced kindled epilepsy and status epilepticus induced spontaneous recurrent seizures. Pentylenetetrazole (40 mg kg⁻¹) (PTZ) administration every second day for a period of 15 days was used to elicit kindled seizure activity in mice. Severity of kindled seizures was assessed in terms of a composite kindled seizure severity score (KSSS). Pilocarpine (100 mg kg⁻¹) was injected every 20 min until the onset of status epilepticus. A spontaneous recurrent seizure severity score (SRSSS) was recorded as a measure of quantitative assessment of the progressive development of spontaneous recurrent seizures induced after pilocarpine status epilepticus. Sub-acute PTZ administration induced the development of severe form of kindled seizures in mice. Further, pharmacological status epilepticus elicited a progressive evolution of spontaneous recurrent seizures in the animals. However, treatment of aminoguanidine, a relatively selective inhibitor of inducible nitric oxide synthase, markedly and dose dependently suppressed the development of both PTZ induced kindled seizures as well as pilocarpine induced spontaneous recurrent seizures. Therefore inducible nitric oxide synthase may be implicated in the development of seizures.     

Structure-Function Investigation of Vsp Serotypes of the Spirochete Borrelia hermsii

Background

Relapsing fever (RF) spirochetes are notable for multiphasic antigenic variation of polymorphic outer membrane lipoproteins, a phenomenon responsible for immune evasion. An additional role in tissue localization is suggested by the finding that isogenic serotypes 1 (Bt1) and 2 (Bt2) of the RF spirochete Borrelia turicatae, which differ only in the Vsp they express, exhibit marked differences in clinical disease severity and tissue localization during infection.

Methodology/Principal Findings

Here we used known vsp DNA sequences encoding for B. turicatae and Borrelia hermsii Vsp proteins with variable regions and then studied whether there are differences in disease expression and tissue localization of their corresponding serotypes during mouse infection. For sequence and structural comparisons we focused exclusively on amino acid residues predicted to project away from the spirochetes surface, referred to as the Vsp dome. Disease severity and tissue localization were studied during persistent infection with individual or mixed serotypes in SCID mice. The results showed that all Vsp domes clustered into 3 main trunks, with the domes for B. turicatae Vsp1 (BtVsp1) and BtVsp2 clustering into separate ones. B. hermsii serotypes whose Vsp domes clustered with the BtVsp1 dome were less virulent but localized to the brain more. The BtVsp2 dome was the oddball among all and Bt2 was the only serotype that caused severe arthritis.

Conclusion/Significance

These findings indicate that there is significant variability in Vsp dome structure, disease severity, and tissue localization among serotypes of B. hermsii.     

Leishmania cell surface prohibitin: role in host–parasite interaction

Proteins selectively upregulated in infective parasitic forms could be critical for disease pathogenesis. A mammalian prohibitin orthologue is upregulated in infective metacyclic promastigotes of Leishmania donovani, a parasite that causes visceral leishmaniasis. Leishmania donovani prohibitin shares 41% similarity with mammalian prohibitin and 95–100% within the genus. Prohibitin is concentrated at the surface of the flagellar and the aflagellar pole, the aflagellar pole being a region through which host–parasite interactions occur. Prohibitin is attached to the membrane through a GPI anchor. Overexpression of wild‐type prohibitin increases protein surface density resulting in parasites with higher infectivity. However, parasites overexpressing a mutant prohibitin with an amino acid substitution at the GPI anchor site to prevent surface expression through GPI‐link show lesser surface expression and lower infective abilities. Furthermore, the presence of anti‐prohibitin antibodies during macrophage–Leishmania interaction in vitro reduces infection. The cognate binding partner for Leishmania prohibitin on the host cell appears to be macrophage surface HSP70, siRNA mediated downregulation of which abrogates the capability of the macrophage to bind to parasites. Leishmania prohibitin is able to generate a strong humoral response in visceral leishmaniasis patients. The above observations suggest that prohibitin plays an important role in events leading to Leishmania–host interaction.     

Modeling metabolic adjustment in <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> upon treatment with isoniazid

Complex biological systems exhibit a property of robustness at all levels of organization. Through different mechanisms, the system tries to sustain stress such as due to starvation or drug exposure. To explore whether reconfiguration of the metabolic networks is used as a means to achieve robustness, we have studied possible metabolic adjustments in Mtb upon exposure to isoniazid (INH), a front-line clinical drug. The redundancy in the genome of M. tuberculosis (Mtb) makes it an attractive system to explore if alternate routes of metabolism exist in the bacterium. While the mechanism of action of INH is well studied, its effect on the overall metabolism is not well characterized. Using flux balance analysis, inhibiting the fluxes flowing through the reactions catalyzed by Rv1484, the target of INH, significantly changes the overall flux profiles. At the pathway level, activation or inactivation of certain pathways distant from the target pathway, are seen. Metabolites such as NADPH are shown to reduce drastically, while fatty acids tend to accumulate. The overall biomass also decreases with increasing inhibition levels. Inhibition studies, pathway level clustering and comparison of the flux profiles with the gene expression data indicate the activation of folate metabolism, ubiquinone metabolism, and metabolism of certain amino acids. This analysis provides insights useful for target identification and designing strategies for combination therapy. Insights gained about the role of individual components of a system and their interactions will also provide a basis for reconstruction of whole systems through synthetic biology approaches.