Sleep fragmentation in mentally retarded people decreases with increasing daylength in spring

We studied the sleep-wake behavior of mentally retarded people from late winter to early summer at 60 degrees N. During this time the daylength increased 8 h 51 min. The data were collected by observing the sleep-wake status of 293 subjects at 20-min intervals for five randomized 24h periods (= recording days). The intervals during which the individual recording days of the same order (1st, 2nd, etc.) were carried out, were called recording periods. Consequently, there were five recording periods, each containing 293 individual recording days. Even though there was overlap among the recording periods, the median daylength from one period to another increased approximately by 100 min. In the initial statistical analysis, the number of wake-sleep transitions was found to differ significantly among the five recording periods (Friedman test, p < 0.001). The mean ranks in the Friedman test suggested that the number of wake-sleep transitions was highest during the 1st and lowest during the 5th recording period. In further statistical analyses using a program for mixed effects regression analysis (MIXOR 2.0) it was found that the increase in daylength during the study period was associated with a simultaneous decrease of approximately 0.5 wake-sleep transitions in the whole study population (p < 0.001). The decrease in the number of wake-sleep transitions was significant only in the subgroups of subjects with a daylength change of more than 350 min between the 1st and 5th recording days (Wilcoxon tests, p < 0.005). This suggests that after a marked prolongation of the natural photoperiod, the reduction in sleep episodes was more probable than after smaller changes in daylength. It is concluded that the sleep of mentally retarded people living in a rehabilitation center at a northern latitude is more fragmented in winter than in early summer and that the change is related probably to the simultaneous increase in the length of the natural photoperiod. The sleep quality of persons living in institutional settings might be improved by increasing the intensity and/or duration of daily artificial light exposure during the darker seasons.     

Transgene insertion induced dominant male sterility and rescue of male fertility using round spermatid injection

Transgene insertions in the mouse often cause mutations at chromosomal loci. Analysis of insertion mutations that cause male sterility may lead to the identification of novel molecular mechanisms implicated in male fertility. Here we show a line of transgenic mice with dominant inheritance of male sterility (DMS) that was found amid several lines that were normally fertile. Transgene-positive males from this line invariably were sterile, whereas transgenic females and transgene-negative male littermates were fertile. Histologic analysis and TUNEL staining for apoptotic cells in DMS testis showed spermatogenesis arrest at metaphase of meiosis I (M-I), accompanied by massive apoptosis of spermatocytes. Meiosis I arrest was incomplete, however, as small numbers of spermatids and spermatozoa were found. Both round spermatids and spermatozoa were evaluated for their permissiveness in the assisted reproductive technologies intracytoplasmic sperm injection (ICSI) and round spermatid injection (ROSI). Surprisingly, ROSI but not ICSI gave live offspring, suggesting that mature sperm had deteriorated by the time of recovery from the epididymis. Mapping the transgene insertion by fluorescence in situ hybridization revealed a site on chromosome 14 D3-E1. Two candidate genes, GFR alpha 2 and GnRH, that were previously mapped to that region and the functions of which in spermatogenesis are well established were not altered in DMS. As a consequence, positional cloning of the DMS locus will be essential to identify new molecules potentially involved in arrest at M-I. Furthermore, mice carrying this genetic trait might be useful for studies of assisted reproductive technologies and male contraceptives.     

Ultrasound stimulates proteoglycan synthesis in bovine primary chondrocytes

Mechanical forces can stimulate the production of extracellular matrix molecules. We tested the efficacy of ultrasound to increase proteoglycan synthesis in bovine primary chondrocytes. The ultrasound-induced temperature rise was measured and its contribution to the synthesis was investigated using bare heat stimulus. Chondrocytes from five cellular isolations were exposed in triplicate to ultrasound (1 MHz, duty cycle 20%, pulse repetition frequency 1 kHz) at average intensity of 580 mW/cm2 for 10 minutes daily for 1-5 days. Temperature evolution was recorded during the sonication and corresponding temperature history was created using a controllable water bath. This exposure profile was used in 10-minute-long heat treatments of chondrocytes. Heat shock protein 70 (Hsp70) levels after one-time treatment to ultrasound and heat was analyzed by Western blotting, and proteoglycan synthesis was evaluated by 35S-sulfate incorporation. Ultrasound treatment did not induce Hsp70, while heat treatment caused a slight heat stress response. Proteoglycan synthesis was increased approximately 2-fold after 3-4 daily ultrasound stimulations, and remained at that level until day 5 in responsive cell isolates. However, chondrocytes from one donor cell isolation out of five remained non-responsive. Heat treatment alone did not increase proteoglycan synthesis. In conclusion, our study confirms that pulsed ultrasound stimulation can induce proteoglycan synthesis in chondrocytes.     

Microfungal species composition and fungal biomass in a coniferous forest soil polluted by alkaline deposition

Isolations of soil microfungi from the humus (F/H-layer) of a coniferous forest soil which was either unpolluted (pH 4.1) or polluted (pH 6.6) for 25 years by deposition of alkaline dust, were made by soil washing and spore plating. Both techniques revealed similar changes in species composition. Alkaline dust exposure caused a reduction in overall species numbers, but led to higher relative isolation frequencies of Mortierella alpina, Oidiodendron tenuissimum, Penicillium montanese, Sagenomella verticillata, and Trichosporiella sporotrichioides. The incidence of M. isabellina, O. cf. clamydosporium, P. spinulosum, Penicillium sp. 1, P. sclerotiorum, Trichoderma viride, and Verticillium bulbillosum was reduced on polluted sites. The amount of the mainly fungal-derived phospholipid fatty acid 18 : 26 decreased by 23%, while the amount of ergosterol increased by 9% in the polluted soil. Offprint requests to: H. Fritze.     

Laminin chains in the basement membranes of human thymus

Summary In recent studies, the α2 chain of laminin (Ln) has been suggested to be the only laminin α chain expressed in mouse and human thymus. We have now used chain-specific monoclonal antibodies and indirect immunofluorescence microscopy to study the expression of laminin chains in samples of foetal and 6-year-old human thymus. The subepithelial basement membrane of the capsule of foetal 16- to 18-week thymus presented a bright immunoreactivity for Ln α1, α3, β1, β3 and γ1 chains but not for α2 chain, suggesting the expression of laminins-1 and-5. Most cortical and medullary epithelial cells, including Hassall's corpuscles, however, lacked laminin immunoreactivity. Immunoreactivity for Ln β2 chain was only seen in basal laminae of larger blood vessels. In thymic specimens from 6-year-old children, immunoreactivity for the laminin α1, α3, β1, β3 and γ1 chains was invariably found in subepithelial basement membrane of the capsule and that for laminin α2 chain was now also distinct but more heterogeneous. Furthermore, the thymic subepithelial basement membrane of the capsule at all stages showed immunore-activity for collagen type VII, forming the anchoring fibres in epithelial basement membranes. The subcapsular thymic epithelium also showed immunoreactivity for the BP 230 antigen and β₄ integrin subunit, both components of hemidesmosomes. The present results show that the thymic subepithelial basement membrane of the capsule presents properties which are commonly seen in stratified and combined epithelia, and are compatible with suggestions of the antigenic similarity of thymic epithelial cells and keratinocytes.     

Pancreatic Duct Obstruction in the Pig: Light Microscopy of Chronic Pancreatitis

Morphological changes of pancreatic tissue in young pigs caused by surgical ligation of the main pancreatic duct are described. Nineteen animals from 6 to 7 weeks in age were operated on and necropsied 3 or 6 to 8 weeks later. Twelve pigs developed a pronounced chronic pancreatitis with complete exocrine insufficiency. Of the 7 animals failing to develop ectasia of pancreatic ducts, 2 died due to surgical complications. In addition, 3 pigs were sham-operated and served as controls. In macroscopical studies it was observed that in the pronounced pancreatitis cases the ligated duct was greatly dilated by a clear watery fluid. Only remnants of pale and firm grandular tissues were seen around the ectatic ducts. Microscopically, typical changes of chronic pancreatitis were noted. Complete disappearance of acini was followed by ductular cell proliferations. Glandular tissues were divided into lobuli by fibrotic tissues and fat cells. The wall of the main pancreatic duct was greatly thickened and fibrotic, presenting intensely proliferating ductular cells and round cell infiltrates. Furthermore, enlarged endocrine islets surrounded by connective tissue fibres were seen.     

Cdk5 regulates the organization of Nestin and its association with p35

The intermediate filament protein nestin is characterized by its specific expression during the development of neuronal and myogenic tissues. We identify nestin as a novel in vivo target for cdk5 and p35 kinase, a critical signaling determinant in development. Two cdk5-specific phosphorylation sites on nestin, Thr-1495 and Thr-316, were established, the latter of which was used as a marker for cdk5-specific phosphorylation in vivo. Ectopic expression of cdk5 and p35 in central nervous system progenitor cells and in myogenic precursor cells induced elevated phosphorylation and reorganization of nestin. The kinetics of nestin expression corresponded to elevated expression and activation of cdk5 during differentiation of myoblast cell cultures and during regeneration of skeletal muscle. In the myoblasts, a disassembly-linked phosphorylation of Thr-316 indicated active phosphorylation of nestin by cdk5. Moreover, cdk5 occurred in physical association with nestin. Inhibition of cdk5 activity-either by transfection with dominant-negative cdk5 or by using a specific cdk5 inhibitor-blocked myoblast differentiation and phosphorylation of nestin at Thr-316, and this inhibition markedly disturbed the organization of nestin. Interestingly, the interaction between p35, the cdk5 activator, and nestin appeared to be regulated by cdk5. In differentiating myoblasts, p35 was not complexed with nestin phosphorylated at Thr-316, and inhibition of cdk5 activity during differentiation induced a marked association of p35 with nestin. These results demonstrate that there is a continuous turnover of cdk5 and p35 activity on a scaffold formed by nestin. This association is likely to affect the organization and operation of both cdk5 and nestin during development.     

Tricholoma matsutake dominates diverse microbial communities in different forest soils

Fungal and actinobacterial communities were analyzed together with soil chemistry and enzyme activities in order to profile the microbial diversity associated with the economically important mushroom Tricholoma matsutake. Samples of mycelium-soil aggregation (shiro) were collected from three experimental sites where sporocarps naturally formed. PCR was used to confirm the presence and absence of matsutake in soil samples. PCR-denaturing gradient gel electrophoresis (DGGE) fingerprinting and direct sequencing were used to identify fungi and actinobacteria in the mineral and organic soil layers separately. Soil enzyme activities and hemicellulotic carbohydrates were analyzed in a productive experimental site. Soil chemistry was investigated in both organic and mineral soil layers at all three experimental sites. Matsutake dominated in the shiro but also coexisted with a high diversity of fungi and actinobacteria. Tomentollopsis sp. in the organic layer above the shiro and Piloderma sp. in the shiro correlated positively with the presence of T. matsutake in all experimental sites. A Thermomonosporaceae bacterium and Nocardia sp. correlated positively with the presence of T. matsutake, and Streptomyces sp. was a common cohabitant in the shiro, although these operational taxonomic units (OTUs) did not occur at all sites. Significantly higher enzyme activity levels were detected in shiro soil. These enzymes are involved in the mobilization of carbon from organic matter decomposition. Matsutake was not associated with a particular soil chemistry compared to that of nearby sites where the fungus does not occur. The presence of a significant hemicellulose pool and the enzymes to degrade it indicates the potential for obtaining carbon from the soil rather than tree roots.     

An automated PCR platform with homogeneous time-resolved fluorescence detection and dry chemistry assay kits

We have developed a novel instrument platform, GenomEra, for small-scale analysis of nucleic acids. The platform combines a rapid thermal cycler, an integrated time-resolved fluorescence measurement unit, and user-friendly software for the analysis of results. Disposable low-cost plastic reaction vessels are designed specifically for the instrument and contain all of the assay-specific reagents in dry form. The appropriate assay protocol is specified on barcodes printed under the vessels and is automatically initiated by the software. Detection is based on the use of sequence-specific probes labeled with intrinsically fluorescent europium or terbium chelates and complementary quencher probes, which enable sensitive, homogeneous closed-tube assays without the risk of carryover contamination. The detection limit of the instrument (background + 3 SD) is approximately 20 pmol/L for both chelates with a dynamic range of nearly four orders of magnitude. The functionality of the platform is demonstrated with a dual-label homogeneous polymerase chain reaction (PCR) assay for the detection of Salmonella using a Magda CA Salmonella assay kit. An internal amplification control is included in each reaction to eliminate false negative results caused by PCR inhibition. Qualitative assay results are automatically interpreted by the software and are available 45 min after sample addition.