Purification of recombinant BtpA and Ycf3, proteins involved in membrane protein biogenesis in Synechocystis PCC 6803

The gene products Ycf3 (hypothetical chloroplast open reading frame) and BtpA (biogenesis of thylakoid protein) are thought to be involved in the biogenesis of the membrane protein complex photosystem I (PSI) from Synechocystis PCC 6803. PSI consists of 12 different subunits and binds more than 100 cofactors, making it a model protein to study different aspects of membrane protein biogenesis. For a detailed biophysical characterization of Ycf3 and BtpA pure proteins must be available in sufficient quantities. Therefore we cloned the corresponding genes into expression vectors. To facilitate purification we created His-tagged versions of Ycf3 and BtpA in addition to the unmodified forms. Immobilized metal affinity chromatography (IMAC) yielded His-tagged proteins which were used for the production of antibodies. Purification strategies for non-tagged proteins could also be established: Ycf3 could be purified in soluble form using a two-step purification in which ammonium sulfate precipitation was combined with anion-exchange chromatography (IEC). BtpA had to be purified from inclusion bodies by two-consecutive IEC steps under denaturing conditions. An optimized refolding protocol was established that yielded pure BtpA. In all cases, MALDI-TOF peptide mass fingerprinting (PMF) was used to confirm protein identity. Initially, size exclusion chromatography and CD-spectroscopy were used for biophysical characterization of the proteins. Both Ycf3 and BtpA show homo-oligomerization in vitro. In summary, purification protocols for Ycf3 and BtpA have been designed that yield pure proteins which can be used to probe the molecular function of these proteins for membrane protein biogenesis.     

The zebrafish van gogh mutation disrupts tbx1, which is involved in the DiGeorge deletion syndrome in humans

The van gogh (vgo) mutant in zebrafish is characterized by defects in the ear, pharyngeal arches and associated structures such as the thymus. We show that vgo is caused by a mutation in tbx1, a member of the large family of T-box genes. tbx1 has been recently suggested to be a major contributor to the cardiovascular defects in DiGeorge deletion syndrome (DGS) in humans, a syndrome in which several neural crest derivatives are affected in the pharyngeal arches. Using cell transplantation studies, we demonstrate that vgo/tbx1 acts cell autonomously in the pharyngeal mesendoderm and influences the development of neural crest-derived cartilages secondarily. Furthermore, we provide evidence for regulatory interactions between vgo/tbx1 and edn1 and hand2, genes that are implicated in the control of pharyngeal arch development and in the etiology of DGS.     

Granulocyte-macrophage-colony stimulating factor in patients with colorectal cancer

Granulocyte-macrophage-colony stimulating factor (GM-CSF) belongs to the group of glycoproteins called colony-stimulating factors (CSFs). It has been shown that the activity of CSFs is not limited to the hematopoietic cells but can also affect the proliferation of colon carcinoma cell lines. The purpose of this investigation was to compare the serum level of GM-CSF in colorectal cancer patients to a control group, to assess the level of GM-CSF in relation to the level of carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9 (CA 19-9), and to define the sensitivity, the specificity and the predictive values of GM-CSF in colorectal cancer. In this study, the serum level of tumour markers was measured in 30 patients with colorectal cancer and in 20 healthy subjects. GM-CSF was assayed using ELISA system, CEA and CA 19-9 were measured by MEIA. The serum levels of CEA, CA 19-9 and GM-CSF were higher in the patients with colorectal cancer than in the control group. The sensitivities of CEA (63%) and CA 19-9 (56%) were lower than the GM-CSF sensitivity (80%). The specificities of tumour markers were 70% (CEA, GM-CSF) and 75% for CA 19-9. The GM-CSF predictive v values were higher than the CEA and CA 19-9 values. These results suggest that GM-CSF may be useful as tumour marker in colorectal cancer, but further studies are needed.     

Energy exploitation in Chirocephalus croaticus (Steuer, 1899) (Crustacea: Anostraca): survival strategy in an intermittent lake

Electron transport system (ETS) activity was measured in Chirocephalus croaticus from the intermittent lake, Petelinjsko Jezero. The ETS activities were measured at 5, 10, 15, 20, and 25 °C, and were studied separately in juveniles, females and males. Juveniles had significantly higher activity than adults at a standard temperature of 20 °C. The mass-specific ETS activity decreased with increasing size of the animals; the value b was 0.787. Respiration rates (R) were determined at 20 °C and the ratio ETS/R (±standard deviation) for C. croaticus was 1.43±0.46 (n=38). ETS activity increased with temperature. Females had higher Q₁₀ than males in higher temperature range (t-test; t=2.50; d.f.=8; p<0.05). Activation energy E_a was higher for females than males (t-test; t=2.35; d.f.=8; p<0.05). Females exhibited lower ETS activity than males over the lower temperature range, but their ETS could function more efficient at higher temperature.     

Suppression of endothelin-converting enzyme-1 during buccal mucosal ulcer healing: effect of chronic alcohol ingestion

Among the factors affecting the efficiency of soft oral tissue healing is endothelin-1 (ET-1), a potent vasoactive peptide produced from a biologically inactive big ET-1 by the action of endothelin-converting enzyme-1 (ECE-1). We investigated the expression of ECE-1 during buccal mucosal ulcer healing in rats maintained for 5 weeks on alcohol containing or control diet. The mucosal activity of ECE-1, characterized by sensitivity to phosphoramidon, was associated with microsomal fraction and showed an elevated (3.1-fold) level in the alcohol diet group. Moreover, the ulcer onset in the alcohol group was reflected in a 39% greater expression of ECE-1 activity, and was accompanied by a 1.4-fold greater increase in TNF-alpha and a 2.5-fold greater enhancement in epithelial cell apoptosis. While in both groups the ulcer healing was associated with a decrease in buccal mucosal expression of ECE-1, as well as a decline in TNF-alpha and apoptosis, the changes were significantly slower in the alcohol diet group and manifested by a 40% delay in healing. Thus, chronic alcohol ingestion leads to up-regulation of ECE-1 expression, induction of TNF-alpha, and triggering apoptotic events that delay the mucosal repair.     

Role of basic fibroblast growth factor in the suppression of apoptotic caspase-3 during chronic gastric ulcer healing.

BACKGROUND: In the course of ulcer healing an array of factors compel mucosal cells to proliferate, differentiate, and migrate to the site of injury. The recognition of triggering cues requires close interaction between the regulatory proteins integrating the growth factor and cytokine- mediated signals that propel cells through the cycle events, or to signal apoptosis. In this study, we analyzed the interplay between mucosal expression of the receptor-bound basic fibroblast growth factor (bFGF-R) and cyclin-dependent kinase (Cdk2), and the activity of apoptotic protease, caspase-3, and constitutive nitric oxide synthase (cNOS) during chronic gastric ulcer healing. METHODS: The experiments were conducted with gastric mucosa of rats at different stages of acetic acid-induced chronic gastric ulcer healing. RESULTS: The ulcer onset (2 days following injury) was characterized by a massive epithelial apoptosis associated with a 33-fold increase in caspase-3 activity and a 7.6-fold drop in cNOS, while the mucosal expression of Cdk2 fell by an 18% and that of bFGF-R by 12%. The ulcer healing was accompanied by a rapid elevation in bFGF-R and Cdk2, and a slow recovery in cNOS activity, while the caspase-3 activity and epithelial apoptosis showed a marked decline. The bFGF-R and Cdk2 reached their maximums of 2.2-2.3-fold at 4-6 day of healing, while the caspase-3 activity and the apoptotic DNA fragmentation showed a 3-fold decline by the 7th day of healing. However, the activity of cNOS remained about 50% lower than that of the controls. CONCLUSIONS: Taken together, these results provide strong indications that the initial phase of ulcer healing involves the inhibition of apoptotic caspase activities by a signaling events initiated by bFGF-receptor activation and propagated by the regulatory kinases that propel the cell cycle progression. Our findings also point towards participation of cNOS in the suppression of proapoptotic activities in gastric mucosa.     

Antimicrobial and Cytotoxic Activity of Extracts of Ferula heuffelii Griseb. ex Heuff. and Its Metabolites

The antimicrobial and cytotoxic activities of isolates (CHCl₃ and MeOH extracts and selected metabolites) obtained from the underground parts of the Balkan endemic plant Ferula heuffelii Griseb . ex Heuff . were assessed. The CHCl₃ and MeOH extracts exhibited moderate antimicrobial activity, being more pronounced against Gram‐positive than Gram‐negative bacteria, especially against Staphylococcus aureus (MIC=12.5 μg/ml for both extracts) and Micrococcus luteus (MIC=50 and 12.5 μg/ml, resp.). Among the tested metabolites, (6E)‐1‐(2,4‐dihydroxyphenyl)‐3,7,11‐trimethyl‐3‐vinyldodeca‐6,10‐dien‐1‐one ( 2 ) and (2S*,3R*)‐2‐[(3E)‐4,8‐dimethylnona‐3,7‐dien‐1‐yl]‐2,3‐dihydro‐7‐hydroxy‐2,3‐dimethylfuro[3,2‐c]coumarin ( 4 ) demonstrated the best antimicrobial activity. Compounds 2 and 4 both strongly inhibited the growth of M. luteus (MIC=11.2 and 5.2 μM , resp.) and Staphylococcus epidermidis (MIC=22.5 and 10.5 μM , resp.) and compound 2 additionally also the growth of Bacillus subtilis (MIC=11.2 μM ). The cytotoxic activity of the isolates was tested against three human cancer cell lines, viz., cervical adenocarcinoma (HeLa), chronic myelogenous leukemia (K562), and breast cancer (MCF‐7) cells. The CHCl₃ extract exhibited strong cytotoxic activity against all cell lines (IC₅₀<11.0 μg/ml). All compounds strongly inhibited the growth of the K562 and HeLa cell lines. Compound 4 exhibited also a strong activity against the MCF‐7 cell line, comparable to that of cisplatin (IC₅₀=22.32±1.32 vs. 18.67±0.75μM ).     

Effects of food quality on trade‐offs among growth,immunity and survival in the greater wax moth Galleria mellonella

The resources available to an individual in any given environment are finite, and variation in life history traits reflect differential allocation of these resources to competing life functions. Nutritional quality of food is of particular importance in these life history decisions. In this study, we tested trade‐offs among growth, immunity and survival in 3 groups of greater wax moth (Galleria mellonella) larvae fed on diets of high and average nutritional quality. We found rapid growth and weak immunity (as measured by encapsulation response) in the larvae of the high‐energy food group. It took longer to develop on food of average nutritional quality. However, encapsulation response was stronger in this group. The larvae grew longer in the low‐energy food group, and had the strongest encapsulation response. We observed the highest survival rates in larvae of the low‐energy food group, while the highest mortality rates were observed in the high‐energy food group. A significant negative correlation between body mass and the strength of encapsulation response was found only in the high‐energy food group revealing significant competition between growth and immunity only at the highest rates of growth. The results of this study help to establish relationships between types of food, its nutritional value and life history traits of G. mellonella larvae.     

Fluctuations of Serum Neuron Specific Enolase and Protein S-100B Concentrations in Relation to the Use of Shunt during Carotid Endarterectomy

Objective

To evaluate the changes in serum neuron specific enolase and protein S-100B, after carotid endarterectomy performed using the conventional technique with routine shunting and patch closure, or eversion technique without the use of shunt.

Materials and Methods

Prospective non-randomized study included 43 patients with severe (>80%) carotid stenosis undergoing carotid endarterectomy in regional anesthesia. Patients were divided into two groups: conventional endarterectomy with routine use of shunt and Dacron patch (csCEA group) and eversion endarterectomy without the use of shunt (eCEA group). Protein S-100B and NSE concentrations were measured from peripheral blood before carotid clamping, after declamping and 24 hours after surgery.

Results

Neurologic examination and brain CT findings on the first postoperative day did not differ from preoperative controls in any patients. In csCEA group, NSE concentrations decreased after declamping (P<0.01), and 24 hours after surgery (P<0.01), while in the eCEA group NSE values slightly increased (P=ns), accounting for a significant difference between groups on the first postoperative day (P=0.006). In both groups S-100B concentrations significantly increased after declamping (P<0.05), returning to near pre-clamp values 24 hours after surgery (P=ns). Sub-group analysis revealed significant decline of serum NSE concentrations in asymptomatic patients shunted during surgery after declamping (P<0.05) and 24 hours after surgery (P<0.01), while no significant changes were noted in non-shunted patients (P=ns). Decrease of NSE serum levels was also found in symptomatic patients operated with the use of shunt on the first postoperative day (P<0.05). Significant increase in NSE serum levels was recorded in non-shunted symptomatic patients 24 hours after surgery (P<0.05).

Conclusion

Variations of NSE concentrations seemed to be influenced by cerebral perfusion alterations, while protein S-100B values were unaffected by shunting strategy. Routine shunting during surgery for symptomatic carotid stenosis may have the potential to prevent postoperative increase of serum NSE levels, a potential marker of brain injury.