Protein stability indicates divergent evolution of PD-(D/E)XK type II restriction endonucleases

Type II restriction endonucleases recognize 4-8 base-pair-long DNA sequences and catalyze their cleavage with remarkable specificity. Crystal structures of the PD-(DE)XK superfamily revealed a common alpha/beta core motif and similar active site. In contrast, these enzymes show little sequence similarity and use different strategies to interact with their substrate DNA. The intriguing question is whether this enzyme family could have evolved from a common origin. In our present work, protein structure stability elements were analyzed and compared in three parts of PD-(DE)XK type II restriction endonucleases: (1) core motif, (2) active-site residues, and (3) residues playing role in DNA recognition. High correlation was found between the active-site residues and those stabilization factors that contribute to preventing structural decay. DNA recognition sites were also observed to participate in stabilization centers. It indicates that recognition motifs and active sites in PD-(DE)XK type II restriction endonucleases should have been evolutionary more conserved than other parts of the structure. Based on this observation it is proposed that PD-(DE)XK type II restriction endonucleases have developed from a common ancestor with divergent evolution.     

Preformed structural elements feature in partner recognition by intrinsically unstructured proteins

Intrinsically unstructured proteins (IUPs) are devoid of extensive structural order but often display signs of local and limited residual structure. To explain their effective functioning, we reasoned that such residual structure can be crucial in their interactions with their structured partner(s) in a way that preformed structural elements presage their final conformational state. To check this assumption, a database of 24 IUPs with known 3D structures in the bound state has been assembled and the distribution of secondary structure elements and backbone torsion angles have been analysed. The high proportion of residues in coil conformation and with phi, psi angles in the disallowed regions of the Ramachandran map compared to the reference set of globular proteins shows that IUPs are not fully ordered even in their bound form. To probe the effect of partner proteins on IUP folding, inherent conformational preferences of IUP sequences have been assessed by secondary structure predictions using the GOR, ALB and PROF algorithms. The accuracy of predicting secondary structure elements of IUPs is similar to that of their partner proteins and is significantly higher than the corresponding values for random sequences. We propose that strong conformational preferences mark regions in IUPs (mostly helices), which correspond to their final structural state, while regions with weak conformational preferences represent flexible linkers between them. In our interpretation, preformed elements could serve as initial contact points, the binding of which facilitates the reeling of the flexible regions onto the template. This finding implies that IUPs draw a functional advantage from preformed structural elements, as they enable their facile, kinetically and energetically less demanding, interaction with their physiological partner.     

19-Nor-1alpha,25-dihydroxyvitamin D2 (paricalcitol) exerts anticancer activity against HL-60 cells in vitro at clinically achievable concentrations

19-Nor-1alpha,25-dihydroxyvitamin D2 (paricalcitol) is an analogue of 1,25(OH)2D3 with reduced calcemic effects that is approved in the United States for the suppression of parathyroid hormone in chronic renal failure. Paricalcitol has anticancer activity in prostate cancer cells. We tested the effects of paricalcitol on the HL-60 leukemia cells, studying cellular differentiation, cell cycle changes, apoptosis and cellular proliferation. Paricalcitol at 10(-8)M concentration induced the maturation of HL-60 cells in a time-dependent manner, as shown by increased expression of CD11b differentiation surface antigen. The ability of HL-60 cells to reduce nitroblue tetrazolium (NBT) was markedly increased after exposure to paricalcitol at 10(-8)M for 72 h. Paricalcitol inhibited colony formation of HL-60 cells in a soft agar semisolid media after 10-day incubation (estimated IC50 of 5 x 10(-9) M. Exposure to 10(-8)M paricalcitol for 72 h increased the number of cells in G0/G1 phase, and decreased the number of cells in S phase, and significantly increased the number of HL-60 cells undergoing apoptosis. The concentration required to achieve inhibition of growth of HL-60 cells is comparable to clinically achievable levels. These findings support the clinical evaluation of paricalcitol as an antileukemia agent.     

Autolysis and aging of Penicillium chrysogenum cultures under carbon starvation: Chitinase production and antifungal effect of allosamidin

In carbon-depleted cultures of Penicillium chrysogenum, age-related chitinases were shown to play a crucial role in both autolysis and fragmentation as indicated by in vivo enzyme inhibition experiments using allosamidin. This pseudotrisaccharide even hindered significantly the outgrowth of new hyphal tips from the surviving yeastlike fragments after glucose supplementation. The antifungal effect of allosamidin on autolyzing P. chrysogenum mycelia was fungistatic rather than fungicidal. In growing hyphae, membrane-bound microsomal chitinase zymogen(s) were detected, which may be indicative of some compartmentalization of these hydrolases. Later, during autolysis, no zymogenic chitinase was detected in any enzyme fraction studied, including microsomes. These observations may explain the different sensitivity of growing and autolyzing mycelia to allosamidin. Chitinases taking part in the age-related fragmentation of hyphae and the outgrowth of surviving hyphal fragments seem to be potent targets for future antifungal drug research.     

Preoperative lymphoscintigraphy guided sentinel lymph node biopsy in malignant melanoma

The authors present preliminary experience with preoperative sentinel lymph node biopsy carried out with lymphoscintigraphy in patients with malignant melanoma. PATIENTS AND METHODS: In the present study patients operated for primary cutaneous malignant melanoma of moderate and high severity were included. On the day of surgery isotope labelled colloid was injected intradermally around the tumor to indicate the lymphatics and to obtain basic information about the localization of the sentinel lymph node(s).During surgery the lymph node(s) previously visualized by the injection of patent-blue staining were detected with the aid of a gamma probe. Simultaneously, the excision of the primary tumor was extended. Histologically verified metastasis in the surgically removed lymph node(s) necessitated block dissection possibly within two weeks. RESULTS: The distribution of patients (19) according to tumor localisation: 2 - upper extremities; 9 - lower extremities; 2 - sacral region; 6 - trunk. Tumor thickness ranged from <1.5 mm (6 patients) to 1.5-3 mm (5 patients) and to >3 mm (8 patients). In two cases the identification of the lymph node has failed. Positive sentinel ymph nodes were detected in two patients. It is noteworthy that with one patient the sentinel lymph node was not regional but intransit. This study was aimed at the development of a suitable method. Further on we wish to try it in prospective randomized studies.     

Improvement of the ecological water qualification system of rivers based on the first results of the Hungarian phytobenthos surveillance monitoring

Results of an ecological quality ratio-based qualification system, developed on the basis of the analysis of 1,161 benthic diatom dataset of the Hungarian national database, are presented herein. Using Kohonen’s Self Organising Map technique, the 25 Hungarian physiographic river types were pooled into six larger distinct categories (diatom river groups). Diatom metrics were tested for their sensitivity to the targeted stressors (nutrients, COD hydromorphological alteration) in each group. The strongest relationships were found in the case of the IPS, SI and TI indices; therefore the average of these metrics (IPSITI) was proposed as a national multimetric index for Hungarian streams. Based on IPSITI values, the ratio of moderate to worse quality water was the highest in those groups containing small rivers. In the case of large, lowland and mid-altitude rivers with fine sediment, the good and moderate ecological status was more characteristic. Applicability of the IPSITI seems to be very useful in case of small- and medium-sized rivers. For these rivers, the index showed a significant relationship with nutrients and organic pollutants. In the case of very large rivers, the stressor–index relationships were not significant because of the insufficient number of samples and the small range of stressors.