Morphological detection and quantification of lipoprotein(a) deposition in atheromatous lesions of human aorta and coronary arteries

Summary Lipoprotein(a), as an atherogenic particle, represents an independent risk factor for coronary heart disease. In the present study the morphological distribution of apoprotein (a) and apoprotein B within the arterial wall is described. Apoprotein B, a constituent of very low-density lipoprotein, low-density lipoprotein and lipoprotein(a) has previously been demonstrated in atheromatous lesions. Lipoprotein(a) possesses an additional protein, designated apoprotein (a). Autopsy material (n=74) from the left coronary artery and from the thoracic aorta has been examined by means of immunohistochemistry and both apoprotein (a) and apoprotein B were detected, primarily associated with the extracellular matrix and accumulating in lesions in the arterial wall. The staining pattern for both antigens was almost always found to be congruent, suggesting that the detection of (a)-antigen has to be attributed at least in part to the presence of lipoprotein(a). It is concluded that both low-density lipoprotein and lipoprotein(a) have an important role in the pathogenesis of atherosclerosis.     

Circulating leucocyte subpopulations in sedentary subjects following graded maximal exercise with hypoxia

Summary Ten healthy sedentary subjects [age, 27.5 (SD 3.5) years; height, 180 (SD 5) cm; mass, 69.3 (SD 6.3) kg] performed two periods of maximal incremental graded cycle ergometer exercise in a supine position. Randomly ordered and using an open spirometric system, one exercise was carried out during normoxia [maximal oxygen consumption ( O_2max)=38.6 (SD 3.5) ml·min^–1·kg^–1; maximal blood lactate concentration, 9.86 (SD 1.85) mmol·l^–1; test duration, 22.6 (SD 2.7) min], the other during hypoxia [ O_2max=33.2 (SD 3.2) ml·min^–1· kg^–1; maximal blood lactate concentration, 10.38 (SD 2.02) mmol·l^–1; test duration, 19.7 (SD 2.8) min]. At rest, immediately (0 p) and 60 min (60 p) after exercise, counts of leucocyte subpopulations (flow cytometry), cortisol and catecholamine concentrations were determined. At 0 p in contrast to normoxia, during hypoxia there was no significant increase of granulocytes. There were no significant differences between normoxia and hypoxia in the increases from rest to 0 p in counts of monocytes, total lymphocytes and lymphocyte subpopulations [clusters of differentiation (CD), CD3⁺, CD4⁺CD45RO^–, CD4⁺CD45RO⁺, CD8⁺CD45RO^–, CD8⁺CD45RO⁺, CD3⁺HLA-DR⁺, CD3^–CD16/CD56⁺, CD3⁺CD16/CD56⁺, CD 19⁺] as well as adrenaline, noradrenaline and cortisol concentrations. The counts of CD3 ^–CD16/CD56⁺-and CD8 ⁺CD45RO⁺-cells increased most. At 60 p, CD3^–CD16/CD56⁺ and CD3⁺CD16/CD56⁺-cell counts were below pre-exercise levels and under hypoxia slightly but significantly lower than under normoxia. We concluded that the exercise-induced mobilization and redistribution of most leucocyte and lymphocyte subpopulations were unimpaired under acute hypoxia at sea level. Reduced increases of granulocyte counts during the study and reduced cell numbers of natural killer cells and cytotoxic, not major histocompatibility complex-restricted T-cells, only indicated marginal effects on the immune system.     

A revised cytoarchitectonic map of the neocortex of the rabbit (Oryctolagus cuniculus)

Summary The cytoarchitectonic parcellation of the rabbit's neocortex has been investigated in 6 hemispheres which had been fixed by perfusion, embedded in paraffin and sectioned at either 9 m or 20 m in various planes. In addition to the classical method of microscopic observation, and automatic scanning procedure using an image analyser for measuring grey level indices was employed. By printing computer plots of various ranges of grey level indices, this method permits visualization of structural differences between various cytoarchitectonic fields. By evaluating the plots, cytoarchitectonic maps can be constructed which are based on objective data and therefore less influenced by subjective judgment than the maps obtained with the classical method. — In some regions the results based on the quantitative method are in agreement with the commonly used maps of Rose (1931), and in other regions widely at variance. It is shown, for instance, that the area striata as defined by Rose (1931) is composed of two distinct fields, viz. areas Oc 1 and 2, which are separated from each other in the rostro-caudal direction. These and other findings are described in detail, compared with the observations of Rose (1931), and related to the literature on functional localization in the rabbit's neocortex. Attention is drawn to the fact that the results obtained in 6 hemispheres leave no doubt that individual variations in size and shape of the entire hemisphere as well as of the various cytoarchitectonic fields do occur, and will have to be taken into account if cytoarchitectonic maps such as those published in the present paper are to be used in the context of experimental work.     

Molecular mechanism of temperature sensing by the circadian clock of Neurospora crassa

Expression levels and ratios of the long (l) and short (s) isoforms of the Neurospora circadian clock protein FREQUENCY (FRQ) are crucial for temperature compensation of circadian rhythms. We show that the ratio of l-FRQ versus s-FRQ is regulated by thermosensitive splicing of intron 6 of frq, a process removing the translation initiation site of l-FRQ. Thermosensitivity is due to inefficient recognition of nonconsensus splice sites at elevated temperature. The temperature-dependent accumulation of FRQ relative to bulk protein is controlled at the level of translation. The 5'-UTR of frq RNA contains six upstream open reading frames (uORFs) that are in nonconsensus context for translation initiation. Thermosensitive trapping of scanning ribosomes at the uORFs leads to reduced translation of the main ORF and allows adjustment of FRQ levels according to ambient temperature.     

Adrenocortical adenoma and carcinoma: histopathological and molecular comparative analysis.

We compared histomorphological features and molecular expression profiles of adrenocortical adenomas (ACAd) and carcinomas (ACCa). A critical histopathological review (mean, 11 slides per patient) was conducted of 37 ACAd and 67 ACCa. Paraffin-embedded tissue cores of ACAd (n = 33) and ACCa (n = 38) were arrayed in triplicate on tissue microarrays. Expression profiles of p53, mdm-2, p21, Bcl-2, cyclin D1, p27, and Ki-67 were investigated by immunohistochemistry and correlated with histopathology and patient outcome using standard statistical methodology. Median follow-up period was 5 years. Tumor necrosis, atypical mitoses, and >1 mitosis per 50 high-power fields were factors that were highly specific for ACCa (P <.001). Number (0 to 4) of unfavorable markers [Ki-67 (+), p21 (+), p27 (+), mdm-2(-)] expressed was significantly associated with mitotic activity and morphologic index (i.e., number of adverse morphologic features) and highly predictive of malignancy (P <.001). Ki-67 overexpression occurred in 0 ACAd and 36% ACCa (P <.001) and was significantly associated with mitotic rate and unfavorable morphologic index (P <.001). Tumor necrosis, atypical mitoses, >5 mitoses per 50 high-power fields, sinusoidal invasion, histologic index of >5, and presence of more than two unfavorable molecular markers were associated significantly with metastasis in ACCa. Well-established histopathologic criteria and Ki-67 can specifically distinguish ACCAd from ACCa. Tumor cell proliferation (Ki-67) correlates with mitotic activity and morphologic index. Tumor morphology is a better predictor of metastatic risk in ACCa than current immunohistochemistry-detected cell cycle regulatory and proliferation-associated proteins.     

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Does a torn anterior cruciate ligament lead to change in the central nervous drive of the knee extensors?

Summary Integrated surface electromyograms of the three superficial parts of the quadriceps and isokinetic knee extensor maximum torque and power production were recorded simultaneously and at different angular velocities in both legs in 11 male subjects with unilateral tear of the anterior cruciate ligament. The cross-sectional area (CSA) of the thigh and its muscular components were measured by computerized tomography. The principal findings were a small but significant decrease in quadriceps CSA on the affected side; a decreased active, but not passive, range of movement; decreased mechanical output, whether or not corrected for differences in CSA; and decreased electrornyographic activity — particularly in rectus femoris. These findings suggest that the reason for the decreased maximum and total knee extensor performance seen in these patients is a change in knee joint receptor afferent inflow.     

A multifunctional bioreactor for three-dimensional cell (co)-culture

Investigation of cell abilities to growth, proliferation and (de)-differentiation in a three-dimensional distribution is an important issue in biotechnological research. Here, we report the development of a new bioreactor for three-dimensional cell culture, which allows for co-cultivation of various cell types with different culture conditions in spatial separation. Preliminary results of neonatal rat cardiomyocyte cultivation are shown. Isolated neonatal rat cardiomyocytes were cultured in spatial separated bioreactor compartments in recirculating medium on a biodegradable fibrin matrix for 2 weeks. Glucose, lactate, and lactate dehydrogenase (LDH), pO2, pCO2, and pH levels were monitored in the recirculated medium, daily. Morphological characterization of matrix and cells was assessed by hematoxylin and eosin staining, and MF-20 co-immunostaining with 4',6-diamidino-2-phenylindole (DAPI). Cell viability was determined by LIVE/DEAD staining before cultivation and on day 3, 7, and 14. The optimized seeding density in the matrix was 2.0 x 10(7) cells retaining cellular proportions over the cell culture period. The bioreactor allows the maintenance of physiologic culture conditions with aerobic cell metabolism (low release of lactate, LDH), a high oxygen tension (pO2-183.7 +/- 18.4 mmHg) and physiological pH values (7.4 +/- 0.02) and a constant level of pCO2 (43.1 +/- 2.9) throughout the experimental course. The cell viability was sufficient after 2 weeks with 82 +/- 6.7% living cells. No significant differences were found between spatial separated bioreactor compartments. Our novel multifunctional bioreactor allows for a three-dimensional culture of cells with spatial separation of the co-cultured cell groups. In preliminary experiments, it provided favorable conditions for the three-dimensional cultivation of cardiomyocytes.     

Dysfunction of axonemal dynein heavy chain Mdnah5 inhibits ependymal flow and reveals a novel mechanism for hydrocephalus formation

Motility of unicellular organisms occurred early in evolution with the emergence of cilia and flagella. In vertebrates, motile cilia are required for numerous functions such as clearance of the airways and determination of left-right body asymmetry. Ependymal cells lining the brain ventricles also carry motile cilia, but their biological function has remained obscure. Here, we show that ependymal cilia generate a laminar flow of cerebrospinal fluid through the cerebral aqueduct, which we term as 'ependymal flow'. The axonemal dynein heavy chain gene Mdnah5 is specifically expressed in ependymal cells, and is essential for ultrastructural and functional integrity of ependymal cilia. In Mdnah5-mutant mice, lack of ependymal flow causes closure of the aqueduct and subsequent formation of triventricular hydrocephalus during early postnatal brain development. The higher incidence of aqueduct stenosis and hydrocephalus formation in patients with ciliary defects proves the relevance of this novel mechanism in humans.