Isolation and characterization of cells from rat adipose tissue developing into adipocytes.

To identify cells developing into adipocytes by accumulation of triglyceride, rat epididymal fat pad cells from small rats were exposed to (3)H-labeled chylomicron fatty acids in vivo and then liberated with collagenase. Tissue remnants were removed by filtration and mature fat cells by flotation. Aggregating cells were then removed by filtration through a 25- micro m nylon screen. Further purification of cells labeled in vivo was obtained by removing floating cells from those adhering to the bottom of a culture dish. The adhering cells multiplied to a confluent monolayer when cultured in Medium 199 containing serum, glucose, insulin, and a triglyceride emulsion. The cells then gradually enlarged due to granulation of the cytoplasm by a lipid-staining material. After about 2 weeks these granules had coalesced forming mature adipocytes of typical signet-ring appearance. Free adipocytes could then be recovered from the cultures by collagenase treatment. After about 2 weeks of culture these cells had the same size (about 30 micro m) as adipocytes recovered in the original collagenase preparation of the rat epididymal fat pad. They contained triglyceride lipase activity and incorporated glucose into triglycerides to the same extent as cells developed in vivo but had higher lipoprotein lipase activity. In vitro, heparin in a low concentration, prostaglandin E(1), isobutylmethylxanthine, and cholera toxin markedly promoted the development of these cells into adipocytes. This could be shown to occur almost completely indicating that this fraction of cells was homogeneous and consisted of cells with the capacity to form adipocytes. The duplication time was about 2 days and did not change with subculturing. Preadipocytes could be obtained by density gradient centrifugation, isolating triglyceride-containing cells either directly from the pad or after 3 days in culture. All of these cells developed into adipocytes as described above but did not multiply as readily. It was concluded that cells from the epididymal fat pad from small rats can be isolated in a homogenous fraction that develops in culture into cells of identical morphology and function as adipocytes formed in vivo. The differentiation of these cells into adipocytes may be manipulated in vitro.     

Escherichia coli ribonucleotide reductase. Radical susceptibility to hydroxyurea is dependent on the regulatory state of the enzyme.

Ribonucleotide reductase catalyzes the reduction of ribonucleotides to their corresponding deoxyribonucleotides via a radical-mediated mechanism. The enzyme from Escherichia coli consists of the two non-identical proteins, R1 and R2, the latter of which contains the necessary free radical located to a tyrosine residue. The radical scavenger hydroxyurea was found to reduce the tyrosyl radical of R2 in a second-order reaction. The rate constant (0.50 M-1 s-1 at 25 degrees C) for this process was several orders of magnitude lower than the hydroxyurea-dependent reduction of free tyrosyl radicals in solution. This difference probably reflects the fact that the R2 tyrosyl radical is buried in the interior of the protein. Formation of the R1R2 complex changed the susceptibility of the radical to hydroxyurea in a manner that reflects the regulatory state of the holoenzyme. Furthermore, binding of substrate or product to the holoenzyme complex made the R2 radical at least 10 times more susceptible to inactivation by hydroxyurea than it was in the isolated R2 protein. One active site mutation in the R1 protein was shown to affect the sensitivity of the tyrosyl radical of R2 differently than wild type protein R1 does. Our results clearly show that the susceptibility of the tyrosyl radical in R2 to inactivation by hydroxyurea can be used as an efficient probe for the regulatory state of the holoenzyme complex.     

Receptor-active glycolipids of epithelial cells of the small intestine of young and adult pigs in relation to susceptibility to infection with Escherichia coli K99

Glycolipids from mucosa scrapings of small intestine of neonatal and adult pigs were tested by the thin-layer chromatogram overlay assay for the binding of Escherichia coli K99. There was practically no binding to acid or non-acid glycolipids of adult pig, known to be resistant to infection with this bacterium. However, piglets, which are susceptible to infection, showed a clear binding to a doublet band in the acid glycolipid fraction. The receptor-active glycolipid was isolated and shown by mass spectrometry, NMR spectroscopy and degradation methods to be NeuGc alpha-3Gal beta 4Glc beta Cer (NeuGc-GM3), the two bands being due to heterogeneity of the ceramide. When tested against various reference glycolipids, NeuAc-GM3 was shown to be inactive. This ganglioside was dominating in adult pig. The apparent developmental disappearance of N-glycolyl groups in glycolipids of intestinal mucosa may have a correspondence in protein-linked sequences as well as thus explain the resistance of adult pigs to infection with E. coli K99.     

Characterization of the binding epitope of the monoclonal antibody DMAb-1 to ganglioside GM2.

Several derivatives of ganglioside GM2 were synthesized for mapping of the binding epitope of a monoclonal antibody raised against this ganglioside. The GM2 ganglioside was modified in both the hydrophobic and the hydrophobilic part of the molecule. The synthesized derivatives were characterized with fast atom bombardment mass spectrometry (FAB-MS). Affinity of the monoclonal antibody for the GM2 derivatives was determined by enzyme-linked immunosorbent assay (ELISA) on microtitre plates or by TLC immunostaining. Modifying the GM2 sialic acid by deacetylation or blocking of the carboxyl moiety abolished the binding to the monoclonal antibody while the cleaving of the glycol group on the sialic acid tail led to a 70% reduced binding affinity. Removal of the fatty acid (lyso-GM2) eliminated the binding to the antibody. GM2 derivatives with fatty acid moieties of 8 carbon atoms or less showed almost no reactivity. GM2 with saturated fatty acids 16:0, 18:0 and 20:0 had binding affinity similar to natural GM2, while the 24:0 fatty acid had only half the binding affinity. The results demonstrate the importance of ganglioside fatty acid composition with regard to ligand binding between the monoclonal antibody and its specific ganglioside antigen. Thus, caution must be shown in the application of immunaffinity methods with monoclonal antibodies for the quantitative determination of glycosphingolipids from different tissues.     

Structure and evolution of the heavy chain from rat immunoglobulin E.

The nucleotide sequence of the rat epsilon-chain mRNA has been determined by sequencing cloned cDNA copies of the mRNA. The established sequence covers the coding region, the 3'-non coding region and most of the 5' non-coding region. A comparison with the nucleotide sequence of the human epsilon-chain constant region reveals that C3 and C4 are the most highly conserved domains. The rat epsilon-chain contains a C-terminal decapeptide which is not present in the human counterpart.     

Crown size and hypodontia in the permanent dentition of moderl Skolt Lapps

The teeth of modern Skolt Lapps from northern Finland are considerably larger than those of their ancestors of the Eighteenth Century. The increase is probably attributable to improved nutrition. One or more teeth, excluding the third molars, were congenitally missing in 18.8% of the population aged 5 to 20 years. Relative to a standard the anterior teeth are larger than the posterior teeth, particularly the premolars. This accords well with the hypodontia pattern which is dominated by premolar agenesis.     

Constituents of human muscle in isometric fatigue.

Three subjects performed five successive isometric contractions to fatigue; the tension in any one experiment was constant at tensions varying from 20 to 80% of the maximal voluntary contraction (MVC). The interval between contractions was held constant at 11 min. Muscle biopsy specimens were obtained at the start of the experiment, after the first, fourth, and fifth, and before the second and fifth of the successive contractions. The concentrations of ATP, CP, glycogen, and lactate were measured in each sample of muscle. Changes in ATP and glycogen were insufficient to be held accountable for the development of isometric fatigue. Changes in CP and lactate were large after fatigue at intermediate tensions, but those of CP were considered unlikely to be responsible for the fatigue. At tensions of 30-50% MVC the increase in lactate could be responsible for fatigue either directly or by indirect changes in pH; at higher and lower tensions the possibility that lactate is directly implicated in the development of fatigue seems remote.     

Proton nuclear magnetic resonance analysis of anomeric structure of glycosphingolipids. The globo-series (one to five sugars).

Proton nuclear magnetic resonance spectroscopy has been reevaluated concerning the assignment of anomeric structure of glycosphingolipids. Solubility problems due to a varying number of sugars are avoided by permethylation, allowing a wide range of glycolipids to be compared. High resolution spectra were recorded in chloroform solution for the following substances with known structure, most of them representing a successive building up of members of the globo-series: ceramide, Galβ1 → 1Cer, a mixture of Glcα1 → 1Cer and Glcβ1 → 1Cer, lactosylceramide, globotriaosylceramide, globotetraosylceramide (globoside), and GalNAcα1 → 3globotetraosylceramide (Forssman hapten). Resonances originating in anomeric protons were identified and possible interference from other signals was defined. A complex set of resonances from H-1 of hexosamines was probably due to two separate conformers of the acetamido group caused by N-methylation. The complexity disappeared upon reduction with LiAlH₄. The chemical shifts and coupling constants were characteristic for the configuration of the glycosidic bond, the type of monomer, and in part for its location in the chain. At present, spectra may be recorded from 200-μg samples. It is concluded that the good quality and resolution obtained make this technique an alternative method to the presently used enzymatic degradation for establishing anomeric structure of glycosphingolipids.