1H- and13C-NMR spectroscopic studies of lipid extracts of the red algaGracilaria longa

Lipid extracts of the red algaGracilaria longa were studied by¹H- and¹³C-NMR spectroscopy. Peaks in the¹³C-NMR spectra attributable to sterols, chlorophylls and carotenoids allowed free and acylated cholesterol, chlorophylla and lutein to be identified as the most abundant components of these classes. A content of 0.5 ± 0.1 μmoles of total cholesterol/g wet alga was estimated from the¹H-NMR spectrum, which also allowed the determination of the phosphatidylcholine/total lipid molar ratio (9.5 ± 0.5%). The¹³C-NMR spectroscopic experiments provided information on the position of the double bonds on the fatty acid residues. A comparison between NMR spectra of lipid extracts obtained for wet and dried alga showed that the alga undergoes both a dramatic peroxidation and some glycolipid degradation during the drying process.     

Matrix genes of measles virus and canine distemper virus: cloning, nucleotide sequences, and deduced amino acid sequences.

The nucleotide sequences encoding the matrix (M) proteins of measles virus (MV) and canine distemper virus (CDV) were determined from cDNA clones containing these genes in their entirety. In both cases, single open reading frames specifying basic proteins of 335 amino acid residues were predicted from the nucleotide sequences. Both viral messages were composed of approximately 1,450 nucleotides and contained 400 nucleotides of presumptive noncoding sequences at their respective 3' ends. MV and CDV M-protein-coding regions were 67% homologous at the nucleotide level and 76% homologous at the amino acid level. Only chance homology was observed in the 400-nucleotide trailer sequences. Comparisons of the M protein sequences of MV and CDV with the sequence reported for Sendai virus (B. M. Blumberg, K. Rose, M. G. Simona, L. Roux, C. Giorgi, and D. Kolakofsky, J. Virol. 52:656-663; Y. Hidaka, T. Kanda, K. Iwasaki, A. Nomoto, T. Shioda, and H. Shibuta, Nucleic Acids Res. 12:7965-7973) indicated the greatest homology among these M proteins in the carboxyterminal third of the molecule. Secondary-structure analyses of this shared region indicated a structurally conserved, hydrophobic sequence which possibly interacted with the lipid bilayer.     

Stereo images of vesicular stomatitis virus assembly.

Viral assembly was studied by viewing platinum replicas of cytoplasmic and outer plasma membrane surfaces of baby hamster kidney cells infected with vesicular stomatitis virus. Replicas of the cytoplasmic surface of the basilar plasma membrane revealed nucleocapsids forming bullet-shaped tight helical coils. The apex of each viral nose cone was anchored to the membrane and was free of uncoiled nucleocapsid, whereas tortuous nucleocapsid was attached to the base of tightly coiled structures. Using immunoelectron microscopy, we identified the nucleocapsid (N) viral protein as a component of both the tight-coil and tortuous nucleocapsids, whereas the matrix (M) protein was found only on tortuous nucleocapsids. The M protein was not found on the membrane. Using immunoreagents specific for the viral glycoprotein (G protein), we found that the amount of G protein per virion varied. The G protein was consistently localized at the apex of viral buds, whereas the density of G protein on the shaft was equivalent to that in the surrounding membrane. These observations suggest that G-protein interaction with the nucleocapsid via its cytoplasmic domain may be necessary for the initiation of viral assembly. Once contact is established, nucleocapsid coiling proceeds with nose cone formation followed by formation of the helical cylinder. M protein may function to induce a nucleocapsid conformation favorable for coiling or may cross-link adjacent turns in the tight coil or both.     

Computer-assisted mathematical analysis of sigmoid biological events

A program in BASIC is described which allows accurate quantificationof some numerical parameters that can be objectively correlatedto biological indexes in sigmoid biological events. Attentionwas focused on the polymerization process of actin (a muscleprotein with a mol. wt of 42 000 daltons) studied as the variationin the OD₃₆₀ index with time. The experimental points, if plotted,can be well approximated by a rational function of the typeOD₃₆₀ = f(t), which passes through the origin and can be representedgraphically by a sigmoid curve. The program was very helpfulin comparing the experimental curves and in analysing significantparameters, such as maximum velocity and asymptote, that characterizethese curves and whose interpretation would otherwise be purelysubjective. Received on July 11, 1985; accepted on January 13, 1986     

Effect of Eimeria falciformis infection on the development of toxoplasmosis in mice

White mice previously infected with 10(2), 10(3) or 10(4) Eimeria falciformis oocysts on days 0, 5, 10 or 30 were inoculated per os with 10(1), 10(2), 10(3) or 10(4) Toxoplasma oocysts. While the results obtained for mice with higher Toxoplasma inocula were consistent, animals with 10(1) and 10(2) oocysts previous inoculation with Eimeria showed important differences related with those infected only with Toxoplasma. For example, survival time was higher in animals infected with both parasites, especially if inoculated with Eimeria 30 days before Toxoplasma infection. Furthermore the number of T. gondii cysts found in the animals previously infected with Eimeria was lower compared with mice inoculated with Toxoplasma only. Body weight of mice infected with Toxoplasma previous infection with Eimeria was almost normal in relation to those infected only with Toxoplasma, indicating a probable pathological effect due to the parasite, more evident in "non immunized" mice.     

HLA-JY328: Mapping studies and expression of a polymorphic HLA class I gene

The JY328 clone was identified in a human genomic library using cDNA corresponding to mRNA for HLA-B7 as a probe. The L/328 cell line was established by cotransformation of mouse Ltk^– cells with the herpes thymidine kinase gene and clone JY328. On Northern blots, RNA from,L/328 strongly hybridized to an HLA class I probe, and an antigen was recognized by an anti-HLA class I framework antibody on the cell surface. A DNA probe corresponding to a segment of intron 7 was developed by comparing the nucleotide sequence of clone JY328 with that of other HLA class I-type genes. Using the radiolabeled probe to screen Southern blots of DNA from families with siblings exhibiting intra-HLA recombinations, a restriction fragment length polymorphism was revealed —a 1.4 kb BstE II band not present in all individuals. A corresponding fragment was apparent in the base sequence of clone JY328. The occurrence of this band on Southern blots established that JY328 maps distinct from and centromeric to the HLA-C locus and near to the HLA-B locus. Antibody absorption studies and cytotoxicity tests indicated that the JY328 gene product was not an HLA-B antigen but that it did specifically absorb CW7-specific antibody. In sum, these results suggest a novel, polymorphic HLA class I gene which expresses a product serologically similar to HLA-Cw7 but which does not map within the corresponding locus.     

Serological discrimination of DR4 haplotypes by radioimmunoassay

Three new alloantigenic specificities of human major histocompatibility complex class 11 molecules have been defined by testing the reactivity of alloantisera at the molecular level. Two of these specificities identify different DR4 haplotypes. The Fe75 specificity is associated with the DR4/DW10 haplotype and the CBC/MRG6 specificity with the DR4/DKT2 haplotype. Both are supertypic specificities and are associated with other DR specificities as well. Both specificities are carried by class 11 molecules belonging to the first DR subset. Together with previously described determinants, these specificities contribute to serological discrimination of the different DR4 haplotypes.     

Mechanism of Excretion of a Bacterial Proteinase: Factors Controlling Accumulation of the Extracellular Proteinase of a Sarcina Strain (Coccus P)

It has been known that the extracellular proteinase of Coccus P is found only in cultures grown in the presence of Ca²⁺. It is now shown that this cation is required neither for synthesis, excretion, or activation of a zymogen nor as a prosthetic factor necessary for enzymatic activity. The only function of Ca²⁺ is to stabilize the active structure of the enzyme molecule, presumably by substituting for absence of S-S bridges. In the absence of Ca²⁺, the excreted proteinase undergoes rapid autodigestion and, instead of the active protein, its hydrolytic products are accumulated in the culture fluid. In minimal medium and under conditions of enzyme stability [presence of Ca²⁺ and Ficoll (Pharmacia)], Coccus P accumulates the proteinase at a gradually reduced speed although the rate of cultural growth remains constant. It is shown that this decline in rate of accumulation is caused by the excreted proteinase itself, possibly acting on its own precursor emerging from the cell in a form susceptible to proteolytic attack and not amenable to Ca²⁺ protection. A proteinase precursor is actually demonstrable in a calciumless culture at the onset of the enzyme accumulation which follows Ca²⁺ addition. It is suggested that excreted proteins require an unfolded (or incompletely folded) structure to cross the cell envelope.     

Osmium tetroxide-zinc iodide reactive sites in the photoreceptor cells of the retina of the rat

Summary The osmium tetroxide-zinc iodide fixative of Champy-Maillet has been used to study the rat's retina at the electron microscope level. Electron opaque deposits were observed all along the photoreceptor cells and concentrated in the outer segments of rods and cones and in the nerve endings. In the outer segments that deposits are located in the inter and intra disk spaces as well as between the disk and outer membranes. In the outer plexiform layer reactive sites include synaptic vesicles and mitochondria; other minor reactive sites are described in the inner segment and inner plexiform layer.Electron opaque deposits were not seen if potassium iodide substitutes zinc iodide in the fixative. However, if osmium tetroxide-potassium iodide fixed retinae are immersed in osmium tetroxide-zinc iodide the characteristic electron-dense material is evidenced at those same sites. The effect of other several fixatives were studied with a similar double fixation procedure. Our finding points to the histochemical demonstration of an unidentified component (s) of the retina which shows a striking specificity of localization and which is made evident when zinc iodide is used in the Champy-Maillet mixture.This work has been supported by grants of the Consejo Nacional de Investigaciones Cientificas y Técnicas, Argentina and U.S. Air Force AF-AFOSR 67-0963 A.We are greatly indebted to Miss Haydée Agoff and to Mr. Alberto Saenz for their skillful technical assistance.