On the effect on specificity of Thr246----Gly mutation in L-lactate dehydrogenase of Bacillus sterothermophilus

The function of the amino acid Thr246 in L-lactate dehydrogenase from Bacillus stearothermophilus has been investigated by site-directed replacement with glycine. Kinetic experiments with a number of 2-oxo acids showed strongly reduced activity for the mutated enzyme. However, the mutant enzyme shows a relative preference for the large hydrophobic sidechains of alpha-keto acids and an even higher specific activity than the wild-type lactate dehydrogenase for the polar oxaloacetate substrate. Graphic analyses indicate that the loss of one hydrogen bond, or intrusion of water into the active site, might be responsible for the reduced activity. The kinetic results suggest that the binding modes of bulky hydrophobic or polar substrates compensate to some degree for the partially disrupted active site.     

Sea star populations diverge by positive selection at a sperm‐egg compatibility locus

Fertilization proteins of marine broadcast spawning species often show signals of positive selection. Among geographically isolated populations, positive selection within populations can lead to differences between them, and may result in reproductive isolation upon secondary contact. Here, we test for positive selection in the reproductive compatibility locus, bindin, in two populations of a sea star on either side of a phylogeographic break. We find evidence for positive selection at codon sites in both populations, which are under neutral or purifying selection in the reciprocal population. The signal of positive selection is stronger and more robust in the population where effective population size is larger and bindin diversity is greater. In addition, we find high variation in coding sequence length caused by large indels at two repetitive domains within the gene, with greater length diversity in the larger population. These findings provide evidence of population‐divergent positive selection in a fertilization compatibility locus, and suggest that sexual selection can lead to reproductive divergence between conspecific marine populations.     

Somatic Mutations in Exocrine Pancreatic Tumors: Association with Patient Survival

KRAS mutations are major factors involved in initiation and maintenance of pancreatic tumors. The impact of different mutations on patient survival has not been clearly defined. We screened tumors from 171 pancreatic cancer patients for mutations in KRAS and CDKN2A genes. Mutations in KRAS were detected in 134 tumors, with 131 in codon 12 and only 3 in codon 61. The GGT>GAT (G12D) was the most frequent mutation and was present in 60% (80/134). Deletions and mutations in CDKN2A were detected in 43 tumors. Analysis showed that KRAS mutations were associated with reduced patient survival in both malignant exocrine and ductal adenocarcinomas (PDAC). Patients with PDACs that had KRAS mutations showed a median survival of 17 months compared to 30 months for those without mutations (log-rank P = 0.07) with a multivariate hazard ratio (HR) of 2.19 (95%CI 1.09–4.42). The patients with G12D mutation showed a median survival of 16 months (log-rank-test P = 0.03) and an associated multivariate HR 2.42 (95%CI 1.14–2.67). Although, the association of survival in PDAC patients with CDKN2A aberrations in tumors was not statistically significant, the sub-group of patients with concomitant KRAS mutations and CDKN2A alterations in tumors were associated with a median survival of 13.5 months compared to 22 months without mutation (log-rank-test P = 0.02) and a corresponding HR of 3.07 (95%CI 1.33–7.10). Our results are indicative of an association between mutational status and survival in PDAC patients, which if confirmed in subsequent studies can have potential clinical application.     

Poly(ADP-ribose) polymerase 1 (PARP-1) binds to 8-oxoguanine-DNA glycosylase (OGG1)

Human 8-oxoguanine-DNA glycosylase (OGG1) plays a major role in the base excision repair pathway by removing 8-oxoguanine base lesions generated by reactive oxygen species. Here we report a novel interaction between OGG1 and Poly(ADP-ribose) polymerase 1 (PARP-1), a DNA-damage sensor protein involved in DNA repair and many other cellular processes. We found that OGG1 binds directly to PARP-1 through the N-terminal region of OGG1, and this interaction is enhanced by oxidative stress. Furthermore, OGG1 binds to PARP-1 through its BRCA1 C-terminal (BRCT) domain. OGG1 stimulated the poly(ADP-ribosyl)ation activity of PARP-1, whereas decreased poly(ADP-ribose) levels were observed in OGG1(-/-) cells compared with wild-type cells in response to DNA damage. Importantly, activated PARP-1 inhibits OGG1. Although the OGG1 polymorphic variant proteins R229Q and S326C bind to PARP-1, these proteins were defective in activating PARP-1. Furthermore, OGG1(-/-) cells were more sensitive to PARP inhibitors alone or in combination with a DNA-damaging agent. These findings indicate that OGG1 binding to PARP-1 plays a functional role in the repair of oxidative DNA damage.     

Novel structural components of the ventral disc and lateral crest in Giardia intestinalis

Giardia intestinalis is a ubiquitous parasitic protist that is the causative agent of giardiasis, one of the most common protozoan diarrheal diseases in the world. Giardia trophozoites attach to the intestinal epithelium using a specialized and elaborate microtubule structure, the ventral disc. Surrounding the ventral disc is a less characterized putatively contractile structure, the lateral crest, which forms a continuous perimeter seal with the substrate. A better understanding of ventral disc and lateral crest structure, conformational dynamics, and biogenesis is critical for understanding the mechanism of giardial attachment to the host. To determine the components comprising the ventral disc and lateral crest, we used shotgun proteomics to identify proteins in a preparation of isolated ventral discs. Candidate disc-associated proteins, or DAPs, were GFP-tagged using a ligation-independent high-throughput cloning method. Based on disc localization, we identified eighteen novel DAPs, which more than doubles the number of known disc-associated proteins. Ten of the novel DAPs are associated with the lateral crest or outer edge of the disc, and are the first confirmed components of this structure. Using Fluorescence Recovery After Photobleaching (FRAP) with representative novel DAP::GFP strains we found that the newly identified DAPs tested did not recover after photobleaching and are therefore structural components of the ventral disc or lateral crest. Functional analyses of the novel DAPs will be central toward understanding the mechanism of ventral disc-mediated attachment and the mechanism of disc biogenesis during cell division. Since attachment of Giardia to the intestine via the ventral disc is essential for pathogenesis, it is possible that some proteins comprising the disc could be potential drug targets if their loss or disruption interfered with disc biogenesis or function, preventing attachment.     

Cleavage of the Phosphodiester Bond of Uridylyl-(3′,5′)-8-Carboxymethylaminoadenosine by Hydronium,Hydroxide and Zinc(Ii) Ions: A Model Study Aimed at Elucidating the Potential of a Carboxylate Function as an Intramolecular Catalyst

Abstract

Uridylyl-(3′,5′)-8-carboxymethylaminoadenosine has been synthesised, and its transesterification to uridine 2′,3′-cyclic phosphate in the presence and absence of Zn²⁺ ion has been studied. The results show that a carboxylate function in the vicinity of the phosphodiester bond accelerates the metal ion promoted cleavage but not the metal ion independent reaction. Under acidic conditions, the predominant reaction is the cleavage of the side chain, giving the 8-amino derivative.     

Spontaneous hypercholesterolemia in cynomolgus monkeys: evidence for defective low-density lipoprotein catabolism.

Spontaneously hypercholesterolemic (SH) cynomolgus monkeys were identified that have average plasma cholesterol of 202 mg/dl, while that in normal monkeys is 119 mg/dl. The LDL from these SH monkeys have lower affinity for fibroblast LDL receptors in vitro. The amount of LDL2 (1.030 mean value of d 1.063 g/ml) required to displace 50% of [125I]LDL was 3.8 micrograms/ml for normal LDL2 and 6.6 micrograms/ml for SH-LDL2. The binding affinity of LDL1 (1.019 mean value of d 1.030 g/ml) was the same in normal and SH animals. LDL turnover experiments showed that the SH monkeys were comprised of two populations. Normal LDL2 was cleared much slower in two of the SH monkeys than in normocholesterolemic animals, suggesting that these two animals have an LDL receptor defect. However, LDL2 isolated from these two SH monkeys was cleared normally in normal monkeys. LDL2 isolated from two other SH monkeys is cleared slower than is normal LDL2 in normal animals, suggesting that these animals have an LDL defect. Thus, the hypercholesterolemia of these SH monkeys is associated with defective LDL catabolism; two animals appear to have functionally defective LDL receptors, and two animals appear to have functionally defective LDL.     

Multi‐gene phylogenetic analyses of New Zealand coralline algae: Corallinapetra Novaezelandiae gen. et sp. nov. and recognition of the Hapalidiales ord. nov.

Coralline red algae from the New Zealand region were investigated in a study focused on documenting regional diversity. We present a multi‐gene analysis using sequence data obtained for four genes (nSSU, psaA, psbA, rbcL) from 68 samples. The study revealed cryptic diversity at both genus and species levels, confirming and providing further evidence of problems with current taxonomic concepts in the Corallinophycidae. In addition, a new genus Corallinapetra novaezelandiae gen. et sp. nov. is erected for material from northern New Zealand. Corallinapetra is excluded from all currently recognized families and orders within the Corallinophycidae and thus represents a previously unrecognized lineage within this subclass. We discuss rank in the Corallinophycidae and propose the order Hapalidiales.     

Muscle use during double poling evaluated by positron emission tomography

Due to the complexity of movement in cross-country skiing (XCS), the muscle activation patterns are not well elucidated. Previous studies have applied surface electromyography (SEMG); however, recent gains in three-dimensional (3D) imaging techniques such as positron emission tomography (PET) have rendered an alternative approach to investigate muscle activation. The purpose of the present study was to examine muscle use during double poling (DP) at two work intensities by use of PET. Eight male subjects performed two 20-min DP bouts on separate days. Work intensity was ～ 53 and 74% of peak oxygen uptake (Vo(2peak)), respectively. During exercise 188 ± 8 MBq of [(18)F]fluorodeoxyglucose ([(18)F]FDG) was injected, and subsequent to exercise a full-body PET scan was conducted. Regions of interest (ROI) were defined within 15 relevant muscles, and a glucose uptake index (GUI) was determined for all ROIs. The muscles that span the shoulder and elbow joints, the abdominal muscles, and hip flexors displayed the greatest GUI during DP. Glucose uptake did not increase significantly from low to high intensity in most upper body muscles; however, an increased GUI (P < 0.05) was seen for the knee flexor (27%) and extensor muscles (16%), and for abdominal muscles (21%). The present data confirm previous findings that muscles of the upper limb are the primary working muscles in DP. The present data further suggest that when exercise intensity increases, the muscles that span the lumbar spine, hip, and knee joints contribute increasingly. Finally, PET provides a promising alternative or supplement to existing methods to assess muscle activation in complex human movements.