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.     

Fractionation of chromatin of liver cell nuclei after mild micrococcal nuclease digestion

Mild micrococcal nuclease treatment of rat and mouse nuclei and fractionation were based on the method of Tata and Baker. Three chromatin fractions, S, P1, P2, were separated, and for each of these fractions the sensitivity to the DNase 1 action was determined. The relative content in these fractions of non-transcribed DNA sequences was established by hydridization with a mouse satellite DNA, and the relative content of transcribed DNA sequences--by hydridization with DNA synthesised on the total poly (A) mRNA. None of the fractions displayed the properties characteristic of active chromatin.     

On the genotoxicity of the pesticides Endodan and Kilacar in 6 different test systems

Two pesticides, the fungicide Endodan (ethylene thiuram monosulphide) and the insecticide-acaricide Kilacar (bis(parachlorophenyl)cyclopropyl methanol), produced or used in the neighbouring countries of Bulgaria and Greece were investigated in a coordinated research programme for their genotoxic effects in a variety of test systems. This included the Ames test, Aspergillus nidulans for mitotic segregation, in vitro human lymphocyte cell cultures for SCE and chromosomal aberrations, in vivo bone marrow cells in hamsters and rats and the dominant lethal test in rats. The genotoxicity of Endodan was found to range from negative to slightly positive in different test systems. At concentrations of 7.5 and 12.0 micrograms/plate together with S9 mix it induced base-pair substitutions in the TA100 strain of Salmonella typhimurium at a rather low level. At a dose of 93 mg/kg b.w. it also caused chromosomal aberrations in acutely treated hamster bone marrow cells. A significant increase of SCE was also found in human lymphocyte cultures at a concentration of 20.0 micrograms/ml. Endodan was found to be negative in A. nidulans for somatic segregation, lymphocyte cultures for chromosomal aberrations and mitotic activity and in rats for dominant lethals and chromosomal aberrations. Kilacar was found to be a weak mutagen in the TA97 strain of S. typhimurium at concentrations of 2.5 and 5.0 micrograms/plate together with S9 mix. At concentrations of 1.0, 1.5 and 2 micrograms/ml Kilacar increased the number of mitotic segregants in A. nidulans by 160%, 220% and 156% respectively over the control. In Syrian hamster bone marrow cells after acute administration at concentrations of 0, 40, 80 and 160 mg/kg, the MI was 5.50, 4.30, 3.10 and 1.30 respectively, and an increase in chromosomal aberrations of about 300% over the control was observed with a concentration of 80 mg/kg. In human lymphocytes no significant changes were observed in either MI or SCE. In the dominant lethal test after chronic treatment of male rats at doses of 5.1, 10.2 and 102.0 mg/kg b.w. no significant mutagenic effect was found although a decrease was shown in the percentage of females with implants mated with treated males in the first week.     

Specificity of Zea mays histone deacetylase is regulated by phosphorylation.

Mono Q ion exchange high performance liquid chromatography (HPLC) reveals that the main histone deacetylase activity (HD1) of germinating Zea mays embryos consists of multiple enzyme forms. Chromatography of HD1 after treatment with alkaline phosphatase yields two distinct histone deacetylase forms (HD1-A, HD1-B). The same is true for chromatography after phosphatase treatment of a total cell extract. One of these enzyme forms (HD1-A) is subject to phosphorylation, which causes a change in the substrate specificity of the enzyme, as shown with HPLC-purified individual core histone species; the substrate specificity for H2A increases more than 2-fold after phosphorylation, whereas the specificity for H3 decreases to about 60%. The total histone deacetylase activity is quantitatively released from isolated nuclei after extraction with moderate ionic strength buffers; no significant residual enzyme activity could be detected in the nuclear matrix.     

Nested PCR for the detection of Aspergillus species in maxillary sinus samples of patients with chronic sinusitis

Background

Fungal rhinosinusitis has become an increasingly recognized disease, being Aspergillus species responsible for most of the cases. Its diagnosis is quite difficult because of the non-specific symptoms and low sensitivity of the current diagnostic methods.

Supplemented nutrition decreases helminth burden and increases drug efficacy in a natural host–helminth system

Gastrointestinal (GI) helminths are common parasites of humans, wildlife, and livestock, causing chronic infections. In humans and wildlife, poor nutrition or limited resources can compromise an individual''s immune response, predisposing them to higher helminth burdens. This relationship has been tested in laboratory models by investigating infection outcomes following reductions of specific nutrients. However, much less is known about how diet supplementation can impact susceptibility to infection, acquisition of immunity, and drug efficacy in natural host–helminth systems. We experimentally supplemented the diet of wood mice (Apodemus sylvaticus) with high-quality nutrition and measured resistance to the common GI nematode Heligmosomoides polygyrus. To test whether diet can enhance immunity to reinfection, we also administered anthelmintic treatment in both natural and captive populations. Supplemented wood mice were more resistant to H. polygyrus infection, cleared worms more efficiently after treatment, avoided a post-treatment infection rebound, produced stronger general and parasite-specific antibody responses, and maintained better body condition. In addition, when applied in conjunction with anthelmintic treatment, supplemented nutrition significantly reduced H. polygyrus transmission potential. These results show the rapid and extensive benefits of a well-balanced diet and have important implications for both disease control and wildlife health under changing environmental conditions.     

Structural damages in adsorbed vaccines affected by freezing

This study was planned to evaluate structural damages in adsorbed vaccines affected by freezing using scanning electron microscopy and X-ray analysis of the elements. Randomly selected 42 vials of eight different types of WHO pre-qualified adsorbed freeze-sensitive vaccines from 10 manufacturers were included in the study. Vaccines were kept at 5 °C. Selected numbers of vials from each type were then exposed to ?25 °C for 24 h periods. All samples were evaluated for their structure using scanning electron microscopy, X-ray analysis of the elements and precipitation time. Scanning electron microscopy of vaccines affected by freezing showed either smooth or rough surfaced conglomerates associated with phosphate content of the precipitate. These vaccines precipitated 2–15 times faster compared to non-frozen samples. Non-frozen samples showed uniform flocculent structure either dense or dispersed. X-ray analysis of precipitates in frozen samples confirmed that the precipitate is mainly aluminium clutters. Scanning electron microscopy confirmed that the lattice structure of bonds between adsorbent and the antigen is broken and aluminium forms conglomerates that grow in size and weight. The precipitation time of vaccines affected by freezing is 4.5 times faster on average compared to non-frozen samples. These facts form the basis of the "shake test".     

The Comparison of the Effects of Three Physiotherapy Techniques on Hamstring Flexibility in Children: A Prospective,Randomized, Single-Blind Study

The aim of the study was to evaluate changes in hamstring flexibility in 120 asymptomatic children who participated in a 6-week program consisting of one physiotherapy session per week and daily home exercises. The recruitment criteria included age (10–13 years), no pain, injury or musculoskeletal disorder throughout the previous year, physical activity limited to school sport. Subjects were randomly assigned to one of the three groups: (1) post-isometric relaxation – PIR (n = 40), (2) static stretch combined with stabilizing exercises – SS (n = 40) and (3) stabilizing exercises – SE (n = 40). Hamstring flexibility was assessed with straight leg raise (SLR), popliteal angle (PA) and finger-to-floor (FTF) tests. The examinations were conducted by blinded observers twice, prior to the program and a week after the last session with the physiotherapist. Twenty-six children who did not participate in all six exercise sessions with physiotherapists were excluded from the analysis. The results obtained by 94 children were analyzed (PIR, n = 32; SS, n = 31; SE, n = 31). In the PIR and SS groups, a significant (P<0.01) increase in SLR, PA, FTF results was observed. In the SE group, a significant (P<0.001) increase was observed in the SLR but not in the PA and FTF (P>0.05). SLR result in the PIR and SS groups was significantly (P<0.001) higher than in the SE group. As far as PA results are concerned, a significant difference was observed only between the SS and SE groups (P = 0.014). There were no significant (P = 0.15) differences regarding FTF results between the three groups. Post-isometric muscle relaxation and static stretch with stabilizing exercises led to a similar increase in hamstring flexibility and trunk forward bend in healthy 10–13-year-old children. The exercises limited to straightening gluteus maximus improved the SLR result, but did not change the PA and FTF results.