Changes of amino acid sequence in PEST-like area and QEEET motif affect degradation rate of D1 polypeptide in photosystem II

The degradation rate of the D1 polypeptide was measured in threeSynechocystis PCC 6803 mutantsin vivo. Mutations were introduced into a putative cleavage area of the D1 polypeptide (QEEET motif) and into the PEST-like area. PEST sequences are often found in proteins with a high turnover rate. The QEEET-motif mutants are CA1 [(E242-E244);Q241H] and E243K, and the third mutation, E229D, was directed to the PEST-like area. During high-light illumination (1500 mol photons m^-2s^-1) that induced photoinhibition of photosystem II (PSII), the half-life time of the D1 polypeptide in mutant E229D (t _1/2=35 min) was about twice as long as in AR (control strain) cells (t _1/2=19 min). In growth light (40 mol photons m^-2s^-1), the degradation rate of the D1 polypeptide in E229D and AR strains was the same (t _1/25 h). In growth light the D1 polypeptide was degraded faster in both QEEET-motif mutants than in the AR strain, but in photoinhibitory light the degradation rates were similar. According to these results, the highly conservative QEEET motif as such is not required for the proteolytic cut of the D1 polypeptide, but it does affect the rate of degradation. No simple correlation existed between the degradation rate of the D1 polypeptide and the susceptibility of PSII to photoinhibition in mutant and AR cells under our experimental conditions.     

Safety issues of Lactobacillus bulgaricus with respect to human gelatinases in vitro

In oral medicine and dentistry probiotics have shown promising results in controlling dental diseases and yeast infections. This study was made to investigate the effect of eight strains of Lactobacillus bulgaricus and their effects on human matrix metalloproteinase-9 (MMP-9). The hypothesis was that these bacteria used in yoghurt production for centuries are not proteolytic and thus can be safely used in the development of probiotic preparations. Bacterial cell fractions and supernatant specimens were prepared and studied with gelatinase zymography and MMP-9 activation was assessed by immunoblotting. The effect of synthetic MMP inhibitors and a serine protease inhibitor (Pefabloc) on bacterial proteinases was studied with zymography. The results showed very low gelatinolytic activity. There was a slight difference between the supernatant and cell fractions so that the supernatant specimens produced weak gelatinolytic bands in zymography while hardly anything was seen in the cell fraction series. The tested synthetic MMP inhibitors and Pefabloc did not affect the proteolytic activity of the lactobacilli strains. The lactobacilli did not seem to induce the conversion of proMMP-9 to its active form. Consequently, our study hypothesis was confirmed and the studied Lactobacillus strains are not likely to degrade host tissue components.     

Non-Employment Histories of Middle-Aged Men and Women Who Died from Alcohol-Related Causes: A Longitudinal Retrospective Study

Background

Long-term patterning of non-employment among problem drinkers is poorly understood. We determined the level and timing of non-employment, and the relative contribution of various types of non-employment among middle-aged persons who died of alcohol-related causes.

Methods

We conducted a longitudinal retrospective register-based study of Finnish men and women aged 45–64 years who died of alcohol-related causes (n = 15 552) or other causes (n = 39 166) in the period 2000–07, or who survived (n = 204 422) until the end of 2007. We traced back the number of days in employment and non-employment for up to 17 years before death or before the end of the study period for the survivors.

Results

The majority (≥56%) of persons who died of alcohol-related causes were in employment up to ten years before death. Over the 17-year period before death, those who died of alcohol-related causes were in employment on average two years less (mean 6.3 years, 95%CI 6.2–6.4) than those dying of other causes (8.2, 8.1–8.3), and five years less than survivors (11.6, 11.5–11.7), when sex and age were adjusted for. The relative role of various types of non-employment differed markedly across the two mortality groups. Among those who died of alcohol-related causes, unemployment accounted for 54% of the total burden of non-employment, in comparison with 29% among those who died of other causes. In contrast, disability pension accounted for 41% of the total burden of non-employment among those who died of alcohol-related causes, but 65% among those who died of other causes.

Conclusions

The results indicate the feasibility of preventing movement out of employment among middle-aged men and women with severe alcohol-related harm, provided that they are identified early on during their working careers and offered effective interventions.     

The community of needle endophytes reflects the current physiological state of Norway spruce

This study investigated fungal endophytes in the needles of Norway spruce (Picea abies) cuttings in relation to host tree growth. We also determined the prevalence of endophytes in needles incubated for six months. The cuttings originated from clonal origins showing slow- and fast-growth in long-term field trials but the heritable differences in growth rate were not yet detected among the studied cutting. Endophytes were isolated from surface-sterilized needles with culture-free DNA techniques. No significant differences were observed between endophyte communities of slow- and fast-growing clonal origins. However, the endophyte community correlated with the current growth rate of cuttings suggesting that endophytes reflect short- rather than long-term performance of a host. The concentration of condensed tannins was similar in slow- and fast-growing clonal origins but it showed a negative relationship with endophyte species richness, implying that these secondary compounds may play an important role in spruce tolerance against fungal infections. More than a third of endophyte species were detected in both fresh and decomposing needles, indicating that many needle endophytes are facultative saprotrophs. Several potentially pathogenic fungal species were also found within the community of saprotrophic endophytes.     

Generation of biologically active endostatin fragments from human collagen XVIII by distinct matrix metalloproteases

Endostatin, a potent inhibitor of endothelial cell proliferation, migration, angiogenesis and tumor growth, is proteolytically cleaved from the C-terminal noncollagenous NC1 domain of type XVIII collagen. We investigated the endostatin formation from human collagen XVIII by several MMPs in vitro. The generation of endostatin fragments differing in molecular size (24-30 kDa) and in N-terminal sequences was identified in the cases of MMP-3, -7, -9, -13 and -20. The cleavage sites were located in the protease-sensitive hinge region between the trimerization and endostatin domains of NC1. MMP-1, -2, -8 and -12 did not show any significant activity against the C-terminus of collagen XVIII. The anti-proliferative effect of the 20-kDa endostatin, three longer endostatin-containing fragments generated in vitro by distinct MMPs and the entire NC1 domain, on bFGF-stimulated human umbilical vein endothelial cells was established. The anti-migratory potential of some of these fragments was also studied. In addition, production of endostatin fragments between 24-30 kDa by human hepatoblastoma cells was shown to be due to MMP action on type XVIII collagen. Our results indicate that certain, especially cancer-related, MMP family members can generate biologically active endostatin-containing polypeptides from collagen XVIII and thus, by releasing endostatin fragments, may participate in the inhibition of endothelial cell proliferation, migration and angiogenesis.     

Reproduction and Pollination in Central European Populations of Silene and Saponaria Species

Flower morphology, flowering phenology, flower visitors, reproductive systems, and stigmatic receptivity of six species of Silene and Saponaria (Silene alba, S. dioica, S. vulgaris, S. nutans, S. noctiflora. Saponaria officinalis) were studied from April to October 1993 and from April to June 1994 in natural populations around Giessen in Hessen/Central Germany and, additionally, in individuals grown from seeds in the Botanical Garden of the University of Giessen. With the exception of Saponaria officinalis and S. noctiflora, all species were regularly visited and pollinated by crepuscular and nocturnal moths and hawkmoths, but only one species, S. alba, was exclusively pollinated by these night-active insects. The other species showed mixed pollination syndromes in which nocturnal and diurnal insects both promoted pollen transfer. Geitonogamy or even autogamy occurred in the gynodioecious and hermaphrodite species S. vulgaris, S. nutans, S. noctiflora, and Saponaria officinalis. Silene noctiflora, the only annual species, is pseudocleistogamous; the majority of its flowers did not open, and fruit set occurred after selfing in bud.     

Reproductive success and genetic divergence among varieties of the rare and endangered <Emphasis Type="Italic">Astragalus cremnophylax</Emphasis> (Fabaceae) from Arizona,USA

Populations of Astragalus cremnophylax from Arizona and related A. humillimus from New Mexico were assessed for morphological, reproductive, genetic, and ecological differences. Results suggest that some of the taxa (i.e., A. cremnophylax var. cremnophylax and A. humillimus) are poor reproducers and environmental fluctuation is not likely a factor contributing to low fecundity in these species. Astragalus cremnophylax var. cremnophylax appears to have low fecundity due to inbreeding depression. Allozyme data from 18 loci show little within-population and taxon genetic variability. Most genetic variability is distributed among populations and taxa. The population of A. cremnophylax var. cremnophylax from the Grand Canyon (South Rim) was genetically depauperate, indicative of the bottleneck caused by historic trampling at this viewpoint. Cape Final, a population of A. cremnophylax var. cremnophylax on the Grand Canyon (North Rim) is genetically different from typical materials of this taxon (South Rim) and is more closely related to other taxa within this species complex. However, the other varieties of A. cremnophylax are more closely related to A. humillimus than the population at Cape Final, suggesting potential species status for Cape Final materials based on allozyme divergence.     

Associations between Trichinella species and host species in Finland

Examination of 627 wild animals--raccoon dogs (Nyctereutes procyonoides), red foxes (Vulpes vulpes), European lynxes (Lynx lynx), brown bears (Ursus arctos), wolves (Canis lupus), and badgers (Meles meles)--revealed Trichinella spp. The prevalence varied according to geographical region of Finland (north; southwest, SW; and southeast, SE) and was the highest among lynxes (70%, SW). The risk of trichinellosis was higher in the SE (odds ratio, OR, 19.4) and SW regions (OR 14.3), as compared with the northern region (OR 1), with no difference between the former 2 regions. Foxes (OR 2.1) and lynxes (OR 1.9) had a higher risk than raccoon dogs (OR 1) of being infected. The distribution of different Trichinella species was evaluated in 87 wild and domestic mammals by multiplex polymerase chain reaction. Trichinella spiralis was detected more often in domestic and synanthropic animals than in sylvatic hosts. Trichinella nativa was detected only in wildlife. Trichinella pseudospiralis was found both in sylvatic and synanthropic hosts. Trichinella britovi was detected only in mixed infections with other Trichinella species. The raccoon dog was the sole host for all 4 Trichinella species and also carried the most intense infections.     

Diffusion-Weighted Imaging in 3.0 Tesla Breast MRI: Diagnostic Performance and Tumor Characterization Using Small Subregions vs. Whole Tumor Regions of Interest

Introduction

Apparent diffusion coefficient (ADC) values are increasingly reported in breast MRI. As there is no standardized method for ADC measurements, we evaluated the effect of the size of region of interest (ROI) to diagnostic utility and correlation to prognostic markers of breast cancer.

Methods

This prospective study was approved by the Institutional Ethics Board; the need for written informed consent for the retrospective analyses of the breast MRIs was waived by the Chair of the Hospital District. We compared diagnostic accuracy of ADC measurements from whole-lesion ROIs (WL-ROIs) to small subregions (S-ROIs) showing the most restricted diffusion and evaluated correlations with prognostic factors in 112 consecutive patients (mean age 56.2±11.6 years, 137 lesions) who underwent 3.0-T breast MRI.

Results

Intra- and interobserver reproducibility were substantial (κ = 0.616–0.784; Intra-Class Correlation 0.589–0.831). In receiver operating characteristics analysis, differentiation between malignant and benign lesions was excellent (area under curve 0.957–0.962, cut-off ADC values for WL-ROIs: 0.87×10⁻³ mm²s^-1; S-ROIs: 0.69×10⁻³ mm²s^-1, P<0.001). WL-ROIs/S-ROIs achieved sensitivities of 95.7%/91.3%, specificities of 89.5%/94.7%, and overall accuracies of 89.8%/94.2%. In S-ROIs, lower ADC values correlated with presence of axillary metastases (P = 0.03), high histological grade (P = 0.006), and worsened Nottingham Prognostic Index Score (P<0.05). In both ROIs, ADC values correlated with progesterone receptors and advanced stage (P<0.01), but not with HER2, estrogen receptors, or Ki-67.

Conclusions

ADC values assist in breast tumor characterization. Small ROIs were more accurate than whole-lesion ROIs and more frequently associated with prognostic factors. Cut-off values differed significantly depending on measurement procedure, which should be recognized when comparing results from the literature. Instead of using a whole lesion covering ROI, a small ROI could be advocated in diffusion-weighted imaging.     

Alport syndrome caused by a 5′ deletion within the COL4A5 gene

Summary Fourteen Italian patients affected with X-linked Alport syndrome were analyzed by Southern blotting, using cDNA probes of the COL4A5 gene. One proband was shown to carry a large deletion (> 38 kb) that included the 5 part of the gene.