Ultrastructural and biological characterization of human choroid cell cultures transformed by Simian Virus 40

A human diploid cell line of choroid origin was isolated from the retrouveal portion of an enucleated eye and designated HC. After 10 passages, when the proliferative capacity of HC cells decreased, they were infected and transformed by Simian Virus 40 (SV40). A proliferating long-term cultured cell line designated HC/SV40 was established and it has been maintained as monolayer for more than 100 passages so far. The two cell lines, HC and HC/SV40, were compared for growth characteristics, capacity to form colonies in soft agar, presence of nuclear T-antigen, and ultrastructure. Cytogenetic analysis was also performed to determine the presence of chromosomal aberrations due to the permanent viral transformation of the cell line. The results indicate that HC/SV40 should be considered the transformed counterparts of HC cells because they are morphologically similar to the latter but can grow in soft agar, possess T-antigen, and show a pattern of karyotypic changes similar to that induced by SV40 in human fibroblasts. The choroid origin of HC and HC/SV40 cell lines was confirmed by the presence, in their cytoplasm, of typical electron dense granules. Their neural origin will make these cell lines very useful for neuropharmacological and differentiation studies.     

Relationship of the replication and incompatibility genes of an IncFVI haemolysin plasmid with other F-like haemolysin plasmids

The replication and incompatibility region of the IncFVI plasmid pSU502 has been isolated by in vitro DNA manipulation as part of a 12.6 kb plasmid, denominated pSU503. Plasmid pSU503 was strongly incompatible with its parental plasmid, pSU1, but was fully compatible with the haemolytic plasmids pSU316 (IncFIII/IV), pHly152 (IncI2) and pSU233 (Inc-pSU233). Furthermore, the 6.9 kb EcoRI fragment of pSU503 which carries the replication and incompatibility determinants of pSU1 did not show any detectable homology (less than 70%) with any of the haemolysin-determining plasmids with which it is compatible. Thus, homologous haemolysin determinants have become linked to apparently unrelated replicons.     

Ecdysteroids in the haemolymph and the ovaries of the firebrat Thermobia domestica (Packard) (Insecta,Thysanura): Correlations with integumental and ovarian cycles

Ecdysteroids were analysed with radioimmunoassay (RIA) and high-performance liquid chromatography (HPLC) in females of the apterygotous insect Thermobia domestica, which ahs overlapping moulting and reproductive cycles. During each moulting cycle, a peak in the haemolymph concentration of ecdysteroids occurs at day 9 (in the 11-day standard cycle), which can be correlated with apolysis and the beginning of new cuticle deposition. The ovaries show a peak of ecdysteroids at day 5 (i.e. one day before egg-laying), which suggests that these hormones are also involved in the reproductive cycle. In both cases, HPLC analysis combined with RIA suggests that the main ecdysteroid is 20-hydroxyecdysone. This duality in the function of ecdysteroids is discussed.     

Comparison of AMP and NADH binding to glycogen phosphorylase b

The binding sites for the allosteric activator, AMP, to glycogen phosphorylase b are described in detail utilizing the more precise knowledge of the native structure obtained from crystallographic restrained least-squares refinement than has hitherto been available. Localized conformational changes are seen at the allosteric effector site that include shifts of between 1 and 2 A for residues Tyr75 and Arg309 and very small shifts for the region of residues 42 to 44 from the symmetry-related subunit. Kinetic studies demonstrate that NADH inhibits the AMP activation of glycogen phosphorylase b. Crystallographic binding studies at 3.5 A resolution show that NADH binds to the same sites on the enzyme as AMP, i.e. the allosteric effector site N, which is close to the subunit-subunit interface, and the nucleoside inhibitor site I, which is some 12 A from the catalytic site. The conformations of NADH at the two sites are different but both conformations are "folded" so that the nicotinamide ring is close (approx. 6 A) to the adenine ring. These conformations are compared with those suggested from solution studies and with the extended conformations observed in the single crystal structure of NAD+ and for NAD bound to dehydrogenases. Possible mechanisms for NADH inhibition of phosphorylase activation are discussed.     

High-resolution proton magnetic resonance studies of the 3'-terminal colicin fragment of 16 S ribosomal RNA from Escherichia coli. Assignment of iminoproton resonances by nuclear Overhauser effect experiments and the influence of adenine dimethylation on the hairpin conformation

The "colicin" fragments comprising the 49 3'-terminal nucleotides of 16 S ribosomal RNA have been isolated from wild-type Escherichia coli and from a kasugamycin-resistant mutant that lacks methylation of two geminal adenine residues. Proton nuclear magnetic resonance (n.m.r.) spectra (500 MHz) were recorded at various temperatures. The low-field resonances arising from the hydrogen-bonded iminoprotons of paired bases were assigned using the nuclear Overhauser effect (n.o.e.). Crucial to the interpretation of the spectra are the resonances that originate from the two hydrogen-bonded iminoprotons of a U X G basepair. Combined with temperature-jump relaxation kinetics experiments the n.o.e.s lead to the conclusion that a conserved A X U/U X G junction in the hairpin is a thermolabile dislocation in the helix. The n.m.r. spectra of the wild-type and mutant fragment are only different with respect to the iminoproton resonances of the two base-pairs adjoining the hairpin loop. The spectra recorded at various temperatures tend to indicate that dimethylation of the adenosines labilizes these base-pairs, but no definitive conclusions are drawn. The results confirm our previous views that dimethylation of the adenosine residues affects the conformation of the hairpin loop.     

The thymic differentiation markers T6 and M241 are two unusual MHC class I antigens

The human beta 2-microglobulin (beta-2m)-associated human thymocyte differentiation antigens T6 and M241 were compared using biochemical techniques. T6 and M241 antigens reside on different molecules with apparent m.w. of 49,000 and 43,000, respectively. Here we show that both proteins have a protein backbone m.w. of 33,000. In addition, T6 and M241 have a large portion of their peptides in common. When we compared the protein backbone m.w. of T6 and M241 with the murine beta-2m-associated thymus leukemia (TL) antigens, we found a considerable difference in size, suggesting that T6 and M241 may not be human homologues of TL antigens and constitute a novel type of major histocompatibility (MHC) class I antigens.     

Stimulation of Free Fatty Acid and Diacylglycerol Accumulation in Cerebrum and Cerebellum During Bicuculline-Induced Status Epilepticus. Effect of Pretreatment with α-Methyl-p-Tyrosine and p-Chlorophenylalanine

The pool size and composition of free fatty acids (FFA) and diglycerides (DG) from the cerebrum and cerebellum of rats undergoing bicuculline-induced seizures were studied. A fourfold increase in cerebral FFA occurred 3-4 min after bicuculline injection; arachidonic and stearic acids were the principal fatty acids accumulated. Cerebellar FFA also increased, but to a lesser extent. An increased production of arachidonic acid took place in the cerebrum as a function of time after bicuculline injection. Other fatty acids produced were oleic, palmitic, and docosahexaenoic acids. A twofold increase in cerebral arachidonic acid was seen at the time of the first generalized tonic-clonic convulsion. However, a 13- to 17-fold increase in arachidonic acid was seen approximately 5-6 min after bicuculline injection. The rise in other FFA was much smaller. Stearoyl- and arachidonoyl-DG were also accumulated. The drug alpha-methyl-p-tyrosine was found to (a) potentiate the bicuculline-stimulated release of cerebellar FFA, and (b) inhibit by 70% the production of stearoyl- and arachidonoyl-DG in the cerebrum and cerebellum. Basal production of FFA was stimulated by p-chlorophenylalanine, but the drug had no effect on the bicuculline-induced changes. Hydrolysis of phospholipids enriched in stearoyl-arachidonoyl groups, such as phosphatidylinositol of excitable membranes, may be stimulated during seizures.     

Effects of water stress on the chlorophyll content,nitrogen level and photosynthesis of leaves of two maize genotypes

The dynamics of leaf chlorophyll level, nitrogen content, photosynthesis and stomatal conductance were followed in detail in two cultivars of maize (Zea mays) during a short period of water stress, applied at tasseling, and during the subsequent recovery phase. Plants used in the experiment were grown in sand-nutrient solution culture under field weather conditions. Water stress reduced chlorophyll levels, stomatal conductance and photosynthesis, but the nitrogen content of the leaves was not affected. It is concluded that the stress-induced loss of chlorophyll is not mediated by a lack of nitrogen. Considerable differences were observed between genotypes in the rate of post-stress recovery of chlorophyll level. Recovery, upon rewatering, of stomatal conductance and photosynthesis preceded that of chlorophyll level. Losses of up to 40% of leaf chlorophyll content were insufficient to affect rates of photosynthesis measured at mid-day.     

Secondary structure features of ribosomal RNA species within intact ribosomal subunits and efficiency of RNA-protein interactions in thermoacidophilic (Caldariella acidophila,Bacillus acidocaldarius) and mesophilic (Escherichia coli) bacteria

Ribosomal subunits of Caldariella acidophila (max.growth temp., 90°C) have been compared to subunits of Bacillus acidocaldarius (max. growth temp., 70°C) and Escherichia coli (max. growth temp., 47°C) with respect to (a) bihelical content of rRNA; (b) G·C content of bihelical domains and (c) tightness of rRNA-protein interactions. The principal results are as follows. 1. Subunits of C. acidophila ribosomes (T_m = 90–93°C) exhibit considerable thermal tolerance over their B. acidocaldarius (T_m = 77°C) and E. coli counterparts (T_m = 72°C). 2. Based on the ‘melting’ hyperchromicities of the intact ribosomal subunits a 51–55% fraction of the nucleotides appears to participate in hydrogen-bonded base pairing regardless of ribosome source, whereas a larger fraction, 67–70%, appears to be involved in hydrogen bonding in the naked rRNA species. 3. The G·C content of bihelical domains of both free and ribosome-bound rRNA increases with increasing thermophily; based on hyperchromicity dispersion spectra of intact subunits and free rRNA, the bihelical parts of C. acidophila rRNA are estimated to contain 63–64% G·C, compared to 58.5% G·C for B. acidocaldarius and 55% G·C for E. coli. 4. The increment in ribosome T_m values with increasing thermophily is greater than the increase in T_m for the free rRNA, indicating that within ribosomes bihelical domains of the thermophile rRNA species are stabilized more efficiently than their mesophile counterparts by proteins or/ and other component(s). 5. The efficiency of the rRNA-protein interactions in the mesophile and thermophile ribosomes has been probed by comparing the releases, with LiCl-urea, of the rRNA species from the corresponding ribosomal subunits stuck to a Celite column through their protein moiety; it has been established that the release of C. acidophila rRNA from the Celite-bound ribosomes occurs at salt-urea concentrations about 4-fold higher than those required to release rRNA from Celite-bound E. coli ribosomes. 6. Compared to E. coli, the C. acidophila 50 and 30 S ribosomal subunits are considerably less susceptible to treatment designed to promote ribosome unfolding through depletion of magnesium ions.