Substructure of membranes in cultivated chick embryo fibroblasts

Summary Electron microscope examination of the plasma membrane of chick embryo fibroblasts cultured in vitro revealed the presence of a single osmiophilic layer about 90 Å thick and a substructure composed of ovoid sub-units associated with an amorphous component. These ovoid sub-units measured approximately 112 Å along the major axis and 75 Å along the minor axis and were composed of a central core, approximately 30 Å by 60 Å, surrounded by a peripheral component.Examination of other membranous components of these cells revealed a similar ovoid subunit structure in a single layered membrane. Differences in thickness and in the sizes of ovoid sub-units were seen in these membranes. The ergastoplasmic membranes, the outer nuclear membranes, the outer mitochondrial and the Golgi membranes were found to be the thinnest.These varied in thickness from approximately 75 Å to 80 Å. The thickest membranes seen were the inner nuclear membranes. These were approximately 100 Å thick. The dimensions of the ovoid sub-units corresponded with differences in the thickness of the various membranes. These findings support the concept of a particulate substructure of cell membranes.This work was aided by Research Grant PH 5593 from the National Science Foundation. Some of the equipment used was purchased with funds from the National Institutes of Health Grant 2TI GM 326. I wish to thank Dr. Robert M. Dougherty from the Department of Microbiology who grew and supplied me with the chick embryo fibroblast cultures used in these studies, and Mrs. Ursula Feller fer her technical assistance.     

IL-6 stimulates osteoclast-like multinucleated cell formation in long term human marrow cultures by inducing IL-1 release

IL-6 enhances the differentiation of pluripotent hematopoietic stem cells but predominantly affects the differentiation of hematopoietic cells in the granulocyte-macrophage lineage. We have previously shown that multinucleated cells (MNC) with many features of the osteoclast phenotype form in long term human marrow cultures. Addition of rhIL-6 (10 to 100 pg/ml) to these cultures significantly increased MNC formation, with greater than 80% of the MNC expressing an Ag that cross-reacts with the mAb 23c6. This antibody preferentially binds to osteoclasts. rhIL-6 did not enhance MNC formation in marrow cultures treated with 1,25 dihydroxyvitamin D3, a potent stimulator of MNC formation, but significantly increased the percentage of MNC that cross-reacted with the 23c6 mAb. Addition of antihuman IL-1 to cultures treated with rhIL-6 totally inhibited the increase in MNC formation stimulated by rhIL-6. In contrast, anti-IL-1 did not affect MNC formation stimulated by 1,25 dihydroxyvitamin D3. Further, conditioned media from marrow cultures exposed to rhIL-6 contained elevated levels of IL-1 beta (500 pg/ml compared to 23 pg/ml in control cultures 15 h after IL-6 addition). These results suggest that the capacity of rhIL-6 to stimulate formation of MNC which cross-react with 23c6 is mediated by induction of release of IL-1 beta.     

Purine metabolism: determination of adenyl deaminase in human lymphocytes

Adenylic acid (AMP) deaminase is a "catabolic enzyme" involved in nucleotide degradation, transforming AMP into inosinic acid (IMP). We present a simple method for the determination of the enzyme activity, which combines high sensitivity with requirement of low quantities of lymphocytes. Human lymphocytes were isolated with a Lymphocyte Separation Medium from FLOW and sonicated. After centrifugation at 2,000 rpm x 10 min and treatment with Norit A, the cells were incubated at 37 degrees C with ATP 0.8 mM and 14C-AMP 0.1 mM (specific activity 12 microCi/mumole) in potassium phosphate 100 mM (pH 7.4). 14C-IMP and 14C-AMP were separated through HPLC by an isocratic elution, with 20 mM KH2PO4 (pH 5.5) at a 1.5 ml/min flow rate. Identification of the nucleotides was carried out through retention time, coelution with internal standards: their evaluation by determining the radioactivity of the collected peaks. The enzyme activity is decreased in patients affected by CLL: the decrease is evident only when data are referred to the single cells and not when they are referred to the protein.     

Unusually high rates of metabolism of DHT in cytosols of the quail uropygial gland

The metabolism of DHT in the cytosol of the quail uropygial gland was found to be so high that the steroid was almost completely inactivated within 2 hours of incubation at 0 C. In these conditions, DHT cannot be used for the characterization of androgen receptors. By contrast, R 1881 and mibolerone, which are not metabolized, can be used as alternative ligands. Moreover, the extremely high metabolism of DHT questions the physiologic role of this steroid in the quail uropygial gland.     

Genetic structure of the population of Sicily.

Genetic heterogeneity within Sicily was investigated on the basis of ACP1, ADA, ESD, GLO1, PGD, PGM1, PGM2, SODA, ABO, and MN gene frequencies, and compared to those of other regions of Italy for which these same loci have been examined. Correspondence analysis revealed no differences within the island, at least at the provincial level, but showed genetic differentiation among Italian regions, distinctly clustering northern, central, and southern populations, respectively. These data indicate a close relationship between Sicily and southern Italy. In addition, the contribution of Middle Eastern populations to the gene pool of Sicily was evident.     

A large deletion in the LDL receptor gene--the cause of familial hypercholesterolemia in three Italian families: a study that dates back to the 17th century (FH-Pavia).

In the LDL-receptor gene, a large rearrangement causing hypercholesterolemia was detected in three apparently unrelated families living in northern Italy. In all probands, binding, internalization, and degradation of 125I-LDL measured in skin fibroblasts were found to be 40%-50% of control values, indicative of heterozygous familial hypercholesterolemia (FH). Southern blot analysis revealed that the probands were heterozygous for a large (25-kb) deletion of the LDL-receptor gene eliminating exons 2-12. The affected subjects possessed two LDL-receptor mRNA species: one of normal size (5.3 kb) and one of smaller size (3.5 kb). In the latter mRNA, the coding sequence of exon 1 is joined to the coding sequence of exon 13, causing a change in the reading frame and thereby giving rise to a premature stop codon. The receptor protein deduced from the sequence of the defective mRNA is a short polypeptide of 29 amino acids, devoid of any function. Tracing these three families back to the 17th century, we found both their common ancestor and the possible origin of the mutation, in a region which is called "Lomellina" and which is located in southwest Lombardy, near the old city of Pavia. Therefore we named the mutation "FH-Pavia."     

Regional and Ontogenic Expression of the NMDA Receptor Subunit NR2D Protein in Rat Brain Using a Subunit-Specific Antibody

Abstract: A polyclonal antibody for the NMDA receptor subunit NR2D has been developed that identifies an ∼160-kDa band on immunoblots from NR2D transfected cells and CNS tissues. No cross-reactivity is seen with other NMDA receptor subunits. The NR2D receptor subunit is N -glycosylated in both brain and transfected cells. Transfected cells expressing NR2D are immunofluorescently labeled, whereas untransfected cells or cells transfected with other NMDA receptor subunit cDNAs are not. Similarly, the NR2D subunit is selectively and quantitatively immunoprecipitated, whereas the NR1, NR2A, or NR2B subunit is not. The relative densities of the NR2D subunit in nine areas of postnatal day 7 and adult rat brains have been determined by quantitative immunoblotting. NR2D was expressed at highest levels in the thalamus, midbrain, medulla, and spinal cord, whereas intermediate levels of this subunit were found in the cortex and hippocampus. Low or undetectable levels were seen in the olfactory bulb, striatum, and cerebellum. Following a peak after the first week of birth, NR2D protein levels decreased by about twofold in adulthood in all rat brain regions examined. More complete ontogenic profiles were determined for the diencephalon, telencephalon, and spinal cord where similar ontogenic patterns were seen. NR2D protein is present at high levels at embryonic stages of development, rises to a peak at postnatal day 7, and decreases but remains measurable during late postnatal life. This study demonstrates the generation and characterization of an antibody selective for the NR2D NMDA receptor subunit as well as a determination of the distribution and ontogenic profile of this subunit in rat brain. The results suggest that native NMDA receptors containing the NR2D subunit may have functional roles not only in the young brain but also in adult brain.     

Form changes in the sea bass, Dicentrarchus labrax (Moronidae: Teleostei), after acclimation to freshwater: an analysis using shape coordinates

Synopsis Morphological changes in the sea bass, Dicentrarchus labrax (Perciformes: Moronidae), were investigated after an experimental acclimation trial to freshwater. The sea bass is an euryhaline species occurring in the Mediterranean and west Atlantic from 30° N to 55° N. Part of the offspring of a pool of breeders was acclimated to freshwater at 9 months of age while maintaining the original stock in marine water. The effect of acclimation to freshwater over the entire form of the fish was studied through geometric morphometrics (shape coordinates). Changes in the form were shown graphically as landmark displacements though age classes 7,12, 15, 19 and 24 months and were discussed in the light of integrated growth. A significantly faster growth in freshwater was detected. Shape coordinates analyzed through multivariate statistics show that significant differences in shape arise after acclimation to freshwater. Moreover, the uniform component of shape change reveals a major effect of stretching or compression perpendicular to the body axis which is common and conservative in both trials. Differences are discussed in terms of selection and ecophenotypism.     

Aerobiological monitoring for fungal spores in a rehabilitation hospital in Northern Italy

In the setting of an aerobiological study of confined environments and environmental prevention of nosocomial infections, aerobiological monitoring of 22 areas in Montescano Medical Rehabilitation Centre (Pavia) was performed in three different months: February, May, and August, 1994. A Lanzoni 3000 spore trap was used for the monitoring. Examination of the results showed that, in the environments monitored, the indoor presence of fungi increased progressively over the three periods considered. The behaviour of individual genera of fungi differed. In the winter periodPenicillium andAspergillus were the most commonly encountered genera.Cladosporium was the dominant genus from the spring to the summer while the yeasts had their maximum counts in the spring morning. The use of air conditioning, combined with the low air exchange between outdoors and indoors in some of the environments certainly played a part in the low concentrations of these latter aerofungi.