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     

Carbon tetrachloride-induced inhibition of hepatocyte lipoprotein secretion: functional impairment of Golgi apparatus in the early phases of such injury

Functional change of liver Golgi apparatus during carbon tetrachloride (CCl4) poisoning was demonstrated both in rat isolated hepatocytes and in the whole animal. The "in vitro" experimental model provided evidence of Golgi derangement early after giving the haloalkane. The "in vivo" analyses also showed that such an alteration involves both formative and secretory sides of the subcellular structure.     

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.     

Analysis of Adenosine Immunoreactivity, Uptake, and Release in Purified Cultures of Developing Chick Embryo Retinal Neurons and Photoreceptors

We have investigated the presence of endogenous adenosine and of mechanisms for adenosine uptake and release in chick embryo retinal neurons and photoreceptors grown in purified cultures in the absence of glial cells. Simultaneous autoradiographic and immunocytochemical analysis showed that endogenous adenosine and the uptake mechanism for this nucleoside colocalize in practically all the photoreceptors, but only in approximately 20% of the neurons. Approximately 25% of the neurons showed either immunocytochemical labeling or autoradiographic labeling, while greater than 50% of the neurons were unlabeled with both techniques. [3H]Adenosine uptake was saturable and could be inhibited by nitrobenzylthioinosine and dipyridamole and by pretreatment of the [3H]adenosine with adenosine deaminase. Although these observations indicate that the uptake is specific for adenosine, only 35% of accumulated radioactivity was associated with adenosine, with the remaining 65% representing inosine, hypoxanthine, and nucleotides plus uric acid. Adenosine as well as several of its metabolites were released by the cells under basal as well as K(+)-stimulated conditions. Potassium-enhanced release was blocked by 10 mM CoCl2 or in Ca2(+)-free, Mg2(+)-rich solutions. The results indicate that retinal cells that synthesize, store, and release adenosine differentiate early during embryogenesis and are therefore consistent with a hypothetical role for adenosine in retinal development.     

Fusion of liposomes and rat brain microsomes examined by two assays

Summary Liposomes are prepared from rat brain microsomal lipid and loaded with either Tb³⁺ or dipicolinic acid (DPA) to test fusion with the Tb-DPA assay. They are also loaded with octadecyl Rhodamine B chloride (R₁₈) to test fusion with the R₁₈ assay. The addition of either Ca²⁺ or Mg²⁺ to loaded liposomes develops fluorescence with both assays. The fluorescence elicited by Mg²⁺ is similar to that elicited by Ca²⁺ if assessed with R₁₈, but much higher if determined by Tb-DPA. The Ca²⁺-dependent fluorescence of the Tb-DPA complex is not suppressed by the addition of EDTA, and therefore it is internal to vesicles. The contrary is true for the Mg²⁺-dependent fluorescence. Rat brain microsomes can be disrupted by adding octylgucoside and reconstituted by removing it by dialysis. We use this procedure to load microsomes with DPA. This allows the use of the Tb-DPA assay for testing the fusion of rat brain microsomes. Reconstituted microsomes fuse with liposomes. This fusion has characteristics similar to those of liposome-liposome fusion. However, no microsome-microsome fusion could be detected with either method. The two methods give different results, owing to the chemical properties of the assays. Indeed Tb-DPA implies the retention of vesicle content, whereas this is not required by the R₁₈ assay.