A Unified Rapid PCR Method for Detection of Normal and Expanded Trinucleotide Alleles of CAG Repeats in Huntington Chorea and CGG Repeats in Fragile X Syndrome

We report on a unified rapid betaine-based-PCR protocol for amplification of the (CAG)n region in Huntington disease (HD) and the (CGG)n region in Fragile X syndrome (FXS), followed by an electrophoretic separation on automated sequencer for precise determination of the triplet numbers. The high betaine concentration (2.5 M betaine) permits precise amplification of the CAG and CGG repeats. Ten HD affected patients and 10 healthy individuals from HD families were re-evaluated. For FXS the CGG region in normal individuals and premutations of about 100 repeats were precisely amplified by this protocol. Ten unrelated FXS premutation carriers and 24 mentally retarded non-FXS affected boys were re-examined by this method. The results totally coincided with the previous ones. This protocol is a good choice as a fast screening test. Within 24 h we can have preliminary information on the patient’s genetic status. Normal individuals, CGG premutation carriers up to 100 repeats, as well as HD patients carrying an expansion up to 50 CAG repeats can be easily clarified. This accounts for a relatively large proportion (about 90%) of the suspected HD and FXS patients, referred to our laboratory for genetic analysis. The calculation of the repeat’s number is more accurate for the correct interpretation of the results, screening tests and genetic counselling.     

Extracellular Neuroactive Amino Acids in the Rat Striatum During Ischaemia: Comparison Between Penumbral Conditions and Ischaemia with Sustained Anoxic Depolarisation

Abstract: Changes in the extracellular levels of excitatory and inhibitory amino acid transmitters were studied in the rat striatum during penumbral ischaemia using intracerebral microdialysis. Effects of penumbral forebrain ischaemia were compared with those of ischaemia with sustained anoxic depolarisation and K⁺ (100 m M ). Comparisons were also made between different groups of animals at 2 and 24 h after dialysis probe implantation. The K⁺ stimulus did not provoke any release of excitatory amino acids in the 24-h group, probably reflecting a decrease of functional synapses adjacent to the probe. During 30 min of penumbral ischaemia, excitatory amino acids did not reach critical concentrations in the extracellular fluid, and increases in levels of inhibitory/modulatory amino acids were similar. On the other hand, severe transient ischaemia resulted in massive synchronous release of many neuroactive excitatory and inhibitory compounds, in both the 2- and 24-h groups. These and other data suggest that changes during severe ischaemia may arise from both neurotransmitter and metabolic pools. It is concluded that is- chaemic damage in the penumbra may not be related to extracellular neuroactive amino acid changes generated within this region.     

Human biliverdin reductase, a previously unknown activator of protein kinase C betaII

Human biliverdin reductase (hBVR), a dual specificity kinase (Ser/Thr/Tyr) is, as protein kinase C (PKC) betaII, activated by insulin and free radicals (Miralem, T., Hu, Z., Torno, M. D., Lelli, K. M., and Maines, M. D. (2005) J. Biol. Chem. 280, 17084-17092; Lerner-Marmarosh, N., Shen, J., Torno, M. D., Kravets, A., Hu, Z., and Maines, M. D. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 7109-7114). Here, by using 293A cells co-transfected with pcDNA3-hBVR and PKC betaII plasmids, we report the co-immunoprecipitation of the proteins and co-purification in the glutathione S-transferase (GST) pulldown assay. hBVR and PKC betaII, but not the reductase and PKC zeta, transphosphorylated in assay systems supportive of activity of only one of the kinases. PKC betaII K371R mutant protein ("kinase-dead") was also a substrate for hBVR. The reductase increased the Vmax but not the apparent Km values of PKC betaII for myelin basic protein; activation was independent of phospholipids and extended to the phosphorylation of S2, a PKC-specific substrate. The increase in substrate phosphorylation was blocked by specific inhibitors of conventional PKCs and attenuated by sihBVR. The effect of the latter could be rescued by subsequent overexpression of hBVR. To a large extent, the activation was a function of the hBVR N-terminal chain of valines and intact ATP-binding site and the cysteine-rich C-terminal segment. The cobalt protoporphyrin-activated hBVR phosphorylated a threonine in a peptide corresponding to the Thr500 in the human PKC betaII activation loop. Neither serine nor threonine residues in peptides corresponding to other phosphorylation sites of the PKC betaII nor PKC zeta activation loop-derived peptides were substrates. The phosphorylation of Thr500 was confirmed by immunoblotting of hBVR.PKC betaII immunocomplex. The potential biological relevance of the hBVR activation of PKC betaII was suggested by the finding that in cells transfected with the PKC betaII, hBVR augmented phorbol myristate acetate-mediated c-fos expression, and infection with sihBVR attenuated the response. Also, in cells overexpressing hBVR and PKC betaII, as well as in untransfected cells, upon treatment with phorbol myristate acetate, the PKC translocated to the plasma membrane and co-localized with hBVR. hBVR activation of PKC betaII underscores its potential function in propagation of signals relayed through PKCs.     

Sequence stratigraphy of Eocene incised valley clastics and associated sediments,Island of Rab,northern Adriatic Sea,Croatia

Clastic sediments of Middle–Late Eocene age were studied on the Island of Rab (northern Adriatic Sea, Croatia) in order to reconstruct their depositional history, depositional environments, and geometry of sandstone bodies. Detailed outcrop logging and mapping revealed the response of depositional systems to frequent relative sea-level changes, which initiated significant basinward and landward shifts of facies, respectively. Tidal sandstones are commonly underlain by shoreface sandstones, and overlain by offshore sandy marls, whereas the latter are again overlain by shoreface sandstones. Major relative sea-level falls initiated basinward shift of depositional systems and the incision of incised valleys or estuaries, and consequently truncated the underlying shelf sediments. In some cases, the accelerated sea-level fall caused rapid shoreface progradation which is interpreted as a forced regression. Relative sea-level rise caused flooding of the incised relief, and deposition of tidal sandstone bodies which overlie type-I sequence boundaries. The coarse lag sediment of these sequence boundaries locally disappears laterally, and the boundary is granulometrically less prominent. All of the major bounding surfaces have been recognized in the sections studied, although the maximum flooding surface is recognized as a thin “zone” instead of a single surface. Altogether, 28 complete sequences, and 15 parasequences are recognized in the informal unit of the Lopar sandstones, documenting the depositional response to high-frequency relative sea-level oscillations. They have so far not been recognized in the Eocene of the eastern Adriatic region.     

Potential Suitability and Viability of Selected Biodiesel Crops in Australian Marginal Agricultural Lands Under Current and Future Climates

The potential environmental suitability and economic viability of growing two biodiesel crops in marginal regions of Australia were explored. Firstly, we used spatial analysis techniques to identify marginal agricultural regions suitable for growing pongam (Pongamia pinnata) and Indian mustard (Brassica juncea), and determined the base socioeconomic viability of investments for the production of biodiesel in the identified areas. Secondly, we used climate change projections (target years 2020 to 2070) from the Commonwealth Scientific, Industrial and Research Organization Mk3.0 global circulation model generated for two emission scenarios (A1B and A1FI) to determine the shift in potential areas for these crops. Under the climate change scenarios tested, the total area suitable for growing pongam between 2040 and 2070 is substantially different from the suitable area under current climate, indicating that long-term investments in this perennial tree crop may not be viable in all regions, especially in southern Australia. There is a greater variation in suitability projections for Indian mustard, although there is more flexibility for cropping options given that it is an annual crop. However, future economic viability is likely to depend on the ability to receive renewable energy certificates for both crops and, in the case of pongam, the certified emission reductions. Opportunities exist for sustainable pongam agroforestry to supply biodiesel to regional towns, cattle stations and mines in northern Australia.     

Axial eye length after retinal detachment surgery

Changes in the eye axial diameter were studied to assess the eye globe impact of conventional operation for retinal detachment. The study included 69 eyes in 69 patients operated on for rhegmatogenous retinal detachment. There were 46.4% of men and 53.6% of women, mean age 52.7 (+/- 15.21) years. Results of preoperative and postoperative ultrasonographic measurement of axial diameter are presented. The mean preoperative and postoperative eye axial diameter was 23.69 (+/- 1.84) mm and 24.43 (+/- 1.91) mm, respectively. Postoperative results showed the axial eye length to increase by a mean of 0.74 (+/- 0.44) mm, yielding a statistically significant difference from the preoperative measurement (p < 0.001). The mean myopia induced by this eyeball elongation was 1.77 D. The encircling band with and without segmental buckling used in surgical repair of retinal detachment creates circular and segmental indentation of the eyeball, thus increasing its axial length. The myopia induced by elongation of the eyeball results in considerable myopia, which requires appropriate correction in the early postoperative period to achieve favorable vision rehabilitation.     

Effectiveness of latanoprost (Xalatan) monotherapy in newly discovered and previously medicamentously treated primary open angle glaucoma patients

We evaluated the effectiveness of latanoprost (Xalatan) monotherapy in primary open angle glaucoma (POAG). Latanoprost is a prostaglandin analogue, the pure 15(R) epimer of 13,14-dihydro-17-phenyl-18,19,20-trinor-PGF2alpha-isopropyl ester. As a prodrug it is being activated by enzymatic hydrolysis in the cornea after which it becomes active acid of latanoprost. Latanoprost is lowering the intraocular pressure (IOP) by increasing the uveoscleral outflow. In this study, latanoprost was used once daily as monotherapy what offers much better compliance for the patients than other combinations of drugs, preserving good IOP control. Based on the significant reduction of the IOP, measured on the day 60 of the trial (mean change in IOP was -5.1 mmHg, with 95% confidence interval in range from -5.6 to -4.5), it is concluded that use of latanoprost is advisable when calculating better IOP control, few side-effects and reductions in costs of potential surgical procedures.     

Influence of psychosocial work-related factors on conventional risk factors of ischemic heart disease and homocysteine in Slovenian male workers

The influence of psychosocial work-related factors on the conventional risk factors of ischemic heart disease (IHD), particularly on the lipid changes and their effect on homocysteine is studied in this paper. Employed males aged 35 to 55 with angina pectoris or a myocardial infarction (IHD group) were compared to a group of individuals without ischemic heart disease (Control Group). Psychosocial factors were assessed using a Swedish Theorell questionnaire. The IHD Group was found to be at a higher risk of IHD due to higher work demands (OR = 1.25), worse job control (OR = 1.23), frequent smoking (OR = 2.2), leadership positions (OR = 3.97), higher BMI (p = 0.059) and higher levels of triglycerides (p = 0.005) and LDL-cholesterol (OR = 1.65). The level of HDL-cholesterol was significantly lower (1.0 vs. 1.4 mmol/L, p < 0.001, OR = 1.64), while the level of C-reactive protein (9.1 vs. 1.8 mg/L) and Interleukin-6 (6.5 vs. 1.6 ng/L) was higher. Homocysteine levels showed borderline significance (p = 0.056). Our study suggests a possible influence of psychosocial work-related factors on IHD risk factors, most of all on low HDL-cholesterol. No connection was found between psychosocial factors and the homocysteine level, shown to be an IHD risk factor at lower levels of approximately 10 micromol/L.     

Radiofrequency ablation as locoregional therapy for unresectable hepatic malignancies: initial results in 24 patients with 5-years follow-up

Radiofrequency ablation (RFA) is one treatment modality for unresectable liver metastases. Patients with hepatic malignancies (n = 24) underwent elective RFA. All tumors were ablated with a curative intent, with a margin of 1 cm, in a single session of RFA. The median diameter of tumor was 3.1 cm (range 1.7-6.9 cm). Studied patients were not candidates for resection due to multifocal hepatic disease, extrahepatic disease, proximity to major vascular structures or presence of cirrhosis with functional hepatic reserve inadequate to tolerate major hepatic resection. Complete tumor necrosis was achieved in 87.5% and tumor recurred in 3 patients (12.5%) with lesions larger than 5 cm. Distant intrahepatic recurrence was diagnosed in another 4 (16.7%). Distant metastases were found in 7 (29.2%) patients. Four of these 7 patients had also distant intrahepatic recurrence of disease. Two and 5-years survival rates were 41.7% (10 patients) and 8.3% (2 patients) respectively. RFA is safe and effective option for patients with unresectable hepatic malignancies smaller than 5 cm without distant metastatic disease. RF ablation resulted in complete tumor necrosis in 87.5% with 2 and 5-years survival rates much higher than with chemotherapy alone or only supportive therapy, when survival is measured in weeks or months. If RFA is unavailable, percutaneous ethanol injection therapy can be done but with inferior survival rates.