Calbindin D(9k): a protein optimized for calcium binding at neutral pH.

The binding of calcium ions by EF-hand proteins depends strongly on the electrostatic interactions between Ca(2+) ions and negatively charged residues of these proteins. We have investigated the pH dependence of the binding of Ca(2+) ions by calbindin D(9k). This protein offers a unique possibility for interpretation of such data since the pK(a) values of all ionizable groups are known. The binding is independent of pH between 7 and 9, where maximum calcium affinity is observed. An abrupt decrease in the binding affinity is observed at pH values below 7. This decrease is due to protonation of acidic groups, leading to modification of protein charges. The pH dependence of the product of the two macroscopic Ca(2+)-binding constants can be formally described by the involvement of two acidic groups with pK(a) = 6.6. Monte Carlo calculations show that the reduction of Ca(2+) binding is strictly determined by variable electrostatic interactions due to pH-dependent changes not only in the binding sites, but also of the overall charge of the protein.     

Reference genes for quantitative reverse transcription-polymerase chain reaction expression studies in wild and cultivated peanut

Background

Molecular genetic studies on rare tumour entities, such as bone tumours, often require the use of decalcified, formalin-fixed, paraffin-embedded tissue (dFFPE) samples. Regardless of which decalcification procedure is used, this introduces a vast breakdown of DNA that precludes the possibility of further molecular genetic testing. We set out to establish a robust protocol that would overcome these intrinsic hurdles for bone tumour research.

Findings

The goal of our study was to establish a protocol, using a modified DNA isolation procedure and quality controls, to select decalcified samples suitable for array-CGH testing. Archival paraffin blocks were obtained from 9 different pathology departments throughout Europe, using different fixation, embedding and decalcification procedures, in order to preclude a bias for certain lab protocols. Isolated DNA samples were subjected to direct chemical labelling and enzymatic labelling systems and were hybridised on a high resolution oligonucleotide chip containing 44,000 reporter elements. Genomic alterations (gains and losses) were readily detected in most of the samples analysed. For example, both homozygous deletions of 0.6 Mb and high level of amplifications of 0.7 Mb were identified.

Conclusions

We established a robust protocol for molecular genetic testing of dFFPE derived DNA, irrespective of fixation, decalcification or sample type used. This approach may greatly facilitate further genetic testing on rare tumour entities where archival decalcified, formalin fixed samples are the only source.     

Phylogeny of the tribe Antirrhineae (Scrophulariaceae) based on morphological andndhF sequence data

Phylogenetic relationships within the tribe Antirrhineae (Scrophulariaceae) are analysed and discussed on the basis of parsimony analyses of morphological andndhF gene sequence data. The results indicate that the tribe Antirrhineae consists of four major groups of genera, theAnarrhinum clade, theGambelia clade, theMaurandya clade, and theAntirrhinum clade. TheAnarrhinum clade, consisting of the Old World bee-pollinated generaAnarrhinum andKickxia, is sister to the rest of the tribe. TheGambelia clade consists of the New World generaGambelia andGalvezia, which are very closely related and pollinated by hummingbirds. TheMaurandya clade consists of one subclade includingMaurandya and a number of related bee- or hummingbird-pollinated New World genera and another subclade with the Old World bee-pollinated generaAsarina andCymbalaria. TheAntirrhinum clade consists mainly of bee-pollinated Old World genera, such asAntirrhinum, Linaria, Chaenorhinum, and their segregates, but also includes the New World generaMohavea andHowelliella, of which the latter is known to be partly pollinated by hummingbirds. It is concluded that hummingbirdpollination has evolved independently within Antirrhineae at least three times from bee-pollinated ancestors.     

Mollugo viscosa sp. nov. (Molluginaceae), a segregate of M. brasiliensis from Minas Gerais in eastern Brazil

The new species Mollugo viscosa Thulin & Harley is described from Minas Gerais in eastern Brazil. It was previously part of the recently published M. brasiliensis Thulin & Harley, a species that was found to be non‐monophyletic in molecular phylogenetic analyses. Mollugo brasiliensis is here divided into a northern species (M. brasiliensis s.str.) in Bahia and a southern species (M. viscosa sp. nov.) in Minas Gerais. The new species differs from M. brasiliensis in its denser and more viscous indumentum, and in its distinctly smaller seeds with smaller tubercles and a smaller hilar peg, and from its sister species M. ulei by being a perennial herb with bisexual flowers.     

Peptidyl-prolyl cis-trans isomerase does not affect the Pro-43 cis-trans isomerization rate in folded calbindin D9k

The calcium-binding protein calbindin D9k has previously been shown to exist in two folded forms only differing in the proline cis-trans isomerism of the Gly-42-Pro-43 amide bond. This bond is located in a flexible loop connecting the two EF-hand Ca2+ sites. Calbindin D9k therefore constitutes a unique test case for investigating if the recently discovered enzyme peptidyl-prolyl cis-trans isomerase (PPIase) can affect the cis-trans exchange rate in a folded protein. The 1H NMR saturation transfer technique has been used to measure the rate of interconversion between the cis and trans forms of calbindin in the presence of PPIase (PPIase:calbindin concentration ratio 1:10) at 35 degrees C. No rate enhancement could be detected.     

Mechanisms and Fitness Costs of Resistance to Antimicrobial Peptides LL-37, CNY100HL and Wheat Germ Histones

Antimicrobial peptides (AMPs) represent a potential new class of antimicrobial drugs with potent and broad-spectrum activities. However, knowledge about the mechanisms and rates of resistance development to AMPs and the resulting effects on fitness and cross-resistance is limited. We isolated antimicrobial peptide (AMP) resistant Salmonella typhimurium LT2 mutants by serially passaging several independent bacterial lineages in progressively increasing concentrations of LL-37, CNY100HL and Wheat Germ Histones. Significant AMP resistance developed in 15/18 independent bacterial lineages. Resistance mutations were identified by whole genome sequencing in two-component signal transduction systems (pmrB and phoP) as well as in the LPS core biosynthesis pathway (waaY, also designated rfaY). In most cases, resistance was associated with a reduced fitness, observed as a decreased growth rate, which was dependent on growth conditions and mutation type. Importantly, mutations in waaY decreased bacterial susceptibility to all tested AMPs and the mutant outcompeted the wild type parental strain at AMP concentrations below the MIC for the wild type. Our data suggests that resistance to antimicrobial peptides can develop rapidly through mechanisms that confer cross-resistance to several AMPs. Importantly, AMP-resistant mutants can have a competitive advantage over the wild type strain at AMP concentrations similar to those found near human epithelial cells. These results suggest that resistant mutants could both be selected de novo and maintained by exposure to our own natural repertoire of defence molecules.     

Cibirhiza spiculata (Apocynaceae), a remarkable new species from eastern Ethiopia

Summary  The new species Cibirhiza spiculata Thulin & Goyder, from Acacia-Commiphora bushland on sand in the Somali National Regional State (Ogaden) of Ethiopia, is described and illustrated. Its conservation status has been assessed. A molecular phylogenetic analysis confirms its placement in the tribe Fockeeae and indicates a position of the new species as sister to C. albersiana. The almost obsolete anther appendages, the outer corona with spreading lobes, as well as the pollinia with apparently single pollen grains, also support its placement in Cibirhiza. However, C. spiculata differs markedly from both previously known species of this genus (C. dhofarensis in Oman and Yemen and C. albersiana in Tanzania and Zambia) by its narrowly linear, subsessile leaves and by the inner corona lobes that are curved inwards over the gynostegium and each ending in a spinulose head.     

Quantification of angiogenesis in estrogen receptor-positive and negative breast carcinoma

Background

The objective of this study was to evaluate angiogenesis according to CD34 antigen expression in estrogen receptor (ER)-positive and negative breast carcinomas.

Methods

This study comprised 64 cases of infiltrating ductal carcinoma in postmenopausal women divided into two groups: Group A: ER-positive, n = 35; and Group B: ER-negative, n = 29. The anti-CD34 monoclonal antibody was used as a marker for endothelial cells. Microvessel count was carried out in 10 fields per slide using a 40× objective lens (magnification 400×). Statistical analysis of the data was performed using Student's t-test (p < 0.05).

Results

The mean number of vessels stained with the anti-CD34 antibody in the estrogen receptor-positive and negative tumors was 23.51 ± 1.15 and 40.24 ± 0.42, respectively. The number of microvessels was significantly greater in the estrogen receptor-negative tumors (p < 0.001).

Conclusion

ER-negative tumors have significantly greater CD34 antigen expression compared to ER-positive tumors.