Monte Carlo simulation studies on the prediction of protein folding types from amino acid composition. In the methodology development for statistical prediction of protein structures, the founders of different methods usually selected different sets of proteins to test their predicted results. Therefore, it is hard to make a fair comparison according to the results they reported. Even if the predictions by different methods are performed for the same set of proteins, there is still such a problem: a method better that the other for one set of proteins would not necessarily remain so when applied to another set of proteins. To tackle this problem, a Monte Carlo simulation method is proposed to establish an objective criterion to measure the accuracy of prediction for the protein folding type. Such an objective accuracy is actually corresponding to the asymptotical limit genereated during the Monte Carlo simulation process. Based on that, it has been found that the average objective accuracy for predicting the all-alpha, all-beta, alpha + beta, and alpha/beta proteins by the least Euclid's distance method (Nakashima, H., K. Nishikawa, and T. Ooi. 1986. J. Biochem. 99:152-162) is 73.0% and that by the least Minkowski's distance method (Chou, P.Y. 1989. Prediction in Protein Structure and the Principles of Protein Conformation. Plenum Press. New York. 549-586) is 70.9%, indicating that the former is better than the latter. However, according to the original reports, the latter claimed a rate of correct prediction with 79.7% but the former with only 70.2%, leading to a completely opposite conclusion. This indicates the necessity of establishing an objective criterion, and a comparison is meaningful only when it is based on the objective criterion. The simulation method and the idea developed here also can be applied to examine any other statistical prediction methods.


Mechanisms of meat batter stabilization: a review. Comminuted meat products are a complex mixture of muscle tissue, solubilized proteins, fat, salt, and water. The two theories that have been presented to explain meat batters stabilization are reviewed. The emulsion theory explains stabilization by the formation of a protein film around fat globules, whereas the physical entrapment theory emphasizes the role of the protein matrix in holding the fat in place during chopping and subsequent heating. However, some aspects of stabilization cannot be explained adequately by either one of these theories. In this article the role of meat proteins, aqueous phase, and lipid phase are examined in light of past and recent research findings.


Food legumes in human nutrition: a personal perspective. Perhaps with the notable exception, and that only in recent years, of red meat, which contributes dietary saturated fats and cholesterol, two well-known reasons in the etiology of heart-related disorders, no single group of foods has been portrayed in such negative terms as the food legumes traditionally have been during the last 50 years of research in food science and human nutrition. Even more alarming are the trends of continued research on such aspects as the deficiency of sulfur-amino acids (both by amino acid analyses as well as rat feeding studies), and the heat lability/stability of proteinase inhibitors and phytohemagglutinins in various legume species. A survey of literature indicates that over 100 research papers were published during the 1981 to 1990 period alone, in just three journals (Journal of Food Science, Journal of Agriculture and Food Chemistry, and Journal of the Science of Food and Agriculture) having the highest citation ratings in food sciences on these three topics, with a general consensus about the facts that were well established as early as the late 1950s. Considering the proliferation of journals publishing food science and human nutrition related work, especially in the Third World countries, the actual number probably would be much higher. This trend also indicates that we are repeating certain aspects of research on the importance of food legumes in human nutrition. Are we really any closer today in our understanding and appreciation of why the nomadic human made such a choice for their very existence during the transition to a more civilized society? This is a high time to project the image of legumes in human nutrition in proper perspective. The validity of our continued research on certain aspects of legumes in human nutrition, at a time when worldwide the research dollars are becoming increasingly harder to come by, is challenged in this review. Essentially, it is a journey through the author's personal diary that raises several questions in justifying the continued research support for at least some nutrition-related work on legumes and an account of what research areas perhaps need to be targeted in the 21st century.


Extrusion of food proteins.

Extrusion of food proteins. Protein extrusion has frustrated earlier predictions regarding its impact in the development of food products. The main reason for this disappointing performance has been its failure to yield fabricated food products with textural quality close enough to that of natural products at competitive prices. Texturized soya protein by extrusion is presently the only commercial success in this area, being incorporated into several convenience products, increasing their protein content and quality and conferring them some desirable sensory properties. Technological and scientific gaps in the extrusion texturization are still to be bridged if this technique is to be applied for upgrading unconventional protein. The precise mechanisms responsible for protein texturization through extrusion are still unclear. Proteins show a very wide range of extrusion behavior that is probably related to large differences in their association properties. New peptide bonds, formed by free amino and carboxylic groups of the protein, were postulated as being responsible for the cross-linking that takes place in protein extrusion. However, disulfide bonds and electrostatic and hydrophobic interactions are regarded presently as the texturization mechanism in this process. The recently suggested suspension (or filled "melt") model for biopolymer extrusion offered a new framework for testing extrusion of novel proteins. According to this view, the large differences between the association properties of proteins produce different types of aggregates. Some of them can be insoluble under extrusion conditions and act as a dispersed phase within the melt phase. The extrusion performance of a protein will thus depend on the amount of insoluble aggregate produced inside the extruder and on protein-protein interactions that occur after the superheated molten mass leaves it.


Examination of the DNA sequence-specific binding properties of heat shock transcription factor in Xenopus laevis embryos. The binding of heat shock transcription factor (HSF) to the heat shock element (HSE) is necessary for transcriptional activation of eukaryotic heat shock protein (HSP) genes. The properties of Xenopus embryo HSF were examined by DNA mobility shift analysis employing a synthetic oligonucleotide corresponding to the proximal HSE in the promoter of the Xenopus HSP70B gene. Heat shock induced activation of HSF binding in Xenopus neurulae was not affected by an inhibition of protein synthesis, indicating that the mode of activation may be posttranslational. Also, while HSF binding was activated in control Drosophila cell extracts by in vitro heat shock or other chemical treatments, HSF binding in Xenopus embryo or somatic cell extract was not. Thus, the activation of Xenopus HSE-HSF binding may occur via a different mechanism compared with Drosophila. Furthermore, we determined that the native size of heat-induced HSF in pre- and post-midblastula stage Xenopus embryos is approximately 530 kilodaltons (kDa), which corresponds to a hexamer made up of 88 kDa monomers. Finally, the slower accumulation of HSP70 mRNA to peak levels found at lower heat shock temperatures was not correlated with HSE-HSF binding activity.


Molecular analysis of different allelic variants of wild-type human p53. The p53 tumour suppressor gene is intensively studied because mutations in this gene are the most common genetic alteration so far identified in human cancer. Considerable emphasis has thus been placed on characterizing the biological differences between mutant and wild-type p53 protein. This has led to the realization that in cultured cells, mutant p53 behaves like an oncogene, whereas wild-type p53 is a tumor suppressor gene. The p53 protein is also a target for the tumour virus oncogene products SV40 large T, adenovirus E1B, and human papillomavirus type 16 E6, which are all capable of forming complexes to the p53 protein. Although p53 represents an extremely important cellular regulatory molecule which is well conserved, there exists two allelic variants of wild-type human p53 that differ both in primary and confirmational structure. One variant contains an arginine at amino acid 72 (p53Arg), whereas the other form contains a proline at this residue (p53Pro). The possible implications for more than one allelic variant of wild-type human p53 in the general population is unknown. The present study was undertaken to compare some of the biological features of the different wild-type p53 variants. We present data demonstrating that there was a post-transcriptional selection against accumulation of both variants of wild-type human p53 in 3T3-A31 cells, arguing that both forms are proliferation inhibitory in these cells. Both variants of human p53 were stabilized by SV40 large T, but did not displace mouse p53 from SV40 large T. Neither allelic variant of human p53 was able to reduce significantly SV40-mediated anchorage-independent growth of 3T3-A31 cells.(ABSTRACT TRUNCATED AT 250 WORDS)


Purification and partial characterization of thiol protease inhibitors from embryos of the brine shrimp Artemia. Thiol protease inhibitor (TPI) proteins in embryos of the brine shrimp Artemia were purified to apparent homogeneity and several of their properties were studied. Four protein fractions containing thiol protease inhibitor activity were obtained by high performance liquid chromatography of Artemia embryo proteins on a C-18 reverse-phase column and these were designated as TPI-1a, -1b, -2, and -3. Acrylamide gel electrophoresis showed that TPI-1a and TPI-1b each consisted of two bands of 11.8 and 13.6 kilodaltons (kDa), while TPI-2 and TPI-3 consisted of only one band of 12.5 kDa. Isoelectric focusing experiments demonstrated that TPI-3 contained one band at pH 5.3, while both TPI-1b and TPI-2 yielded bands at pH 5.2 and 5.3. TPI-1a did not yield any major bands. Amino acid composition analyses of the Artemia TPI proteins showed them to be remarkably similar to one another. All were rich in valine and aspartic and glutamic acids, and devoid of cysteine. Partial trypsin digestion of TPI-1b, TPI-2, and TPI-3 yielded several peptides with identical mobilities on a reverse-phase column and several other peptides with different mobilities, suggesting that the multiple forms of Artemia TPIs may have originated from the same parental protein. N-terminal amino acid sequence analyses of TPI-3 suggest that Artemia TPI proteins are members of the type I cystatin family of protease inhibitors.


A novel 49-kilodalton protein from Artemia cross-links microtubules in vitro. A 49 kilodalton (kDa) protein, previously proposed to cross-link microtubules, was purified to apparent homogeneity from cell-free extracts of the brine shrimp Artemia. When incubated with tubulin under assembly conditions, the purified 49-kDa protein cross-linked the resulting microtubules. Preformed microtubules were also cross-linked when incubated with the 49-kDa protein. Upon centrifugation through sucrose cushions the 49-kDa protein cosedimented with microtubules, suggesting a stable association between the cross-linking protein and tubulin. Such microtubules were interconnected by particles which were circular, bilobed, or elongated in shape. Disruption of microtubule cross-linking and dissociation of the 49-kDa protein from microtubules occurred in the presence of ATP and 5'-adenylyl-imidodiphosphate (AMP-PNP), a nonhydrolyzable analogue of ATP. The 49-kDa protein was moderately resistant to heat, it did not stimulate tubulin assembly, and it did not react with antibodies to neural microtubule-associated proteins (MAPs) and kinesin. These observations indicate that the 49-kDa protein is different from many known MAPs, a conclusion strengthened by the inability of antibodies raised to the 49-kDa protein to recognize these proteins. The amino terminal 15 amino acid residues of the 49-kDa protein were determined by Edman digestion and an antibody raised to this peptide reacted with the 49-kDa protein on Western blots. Microtubule cross-linking was unaffected by the synthetic amino-terminal peptide, even when it was present at a fivefold molar excess over the 49-kDa protein. A search of three protein databanks revealed that the amino terminus of the 49-kDa protein is unique among published sequences.(ABSTRACT TRUNCATED AT 250 WORDS)


Mutational analysis of a DNA sequence involved in linking gene expression to the cell cycle. Entry of budding yeast cells into the mitotic cell cycle requires the activity of a conserved regulatory kinase encoded by the CDC28 gene. The kinase is thought to trigger entry into the cell cycle or START, through association with a number of regulatory subunits known as G1 cyclins. A number of genes whose transcription is dependent on CDC28 and thus linked to START are controlled by two transcription factors, SWI4 and SWI6. The genes controlled by SWI4 and SWI6 include two known G1 cyclins (CLN1 and CLN2), a putative new G1 cyclin (HCS26), and the HO gene whose product initiates cell type switching. SWI4 and SWI6 act through a repeated sequence element, SCB (SWI4,6-dependent cell cycle box), found 2-10 times in the upstream regulatory sequences of target genes. We have constructed a library of mutants in the SCB using doped oligonucleotide mutagenesis. All single base pair changes examined compromised the ability of the SCB to activate transcription in vivo. Analysis of the behaviour of the mutant SCBs in an in vitro DNA binding assay shows that the inability to activate transcription can be explained by reduced binding of SWI4 and SWI6 to the mutant SCBs. This analysis, together with a consideration of the SCBs found upstream of known SWI4,6-dependent genes, leads to the proposal of a revised consensus sequence for this important regulatory element.


Regulation of p105wee1 and p34cdc2 during meiosis in Schizosaccharomyces pombe. Temperature-sensitive pat1 mutants of the fission yeast Schizosaccharomyces pombe can be induced to undergo meiosis at the restrictive temperature, irrespective of the mat1 configuration and the nutritional conditions. Using a combination of exit from stationary phase and thermal inactivation of the 52-kilodalton protein kinase that is encoded by the pat1 (also called ran1) gene, highly synchronous meiotic cultures were obtained. Synthesis and tyrosyl phosphorylation of p34cdc2 was evident during meiotic G1 and S phases. During this period there was increased expression of p105wee1, a protein kinase implicated in the tyrosyl phosphorylation of p34cdc2. Following a relatively brief G2 period, during which a reduction in the steady-state level of p105wee1 occurred, there was an approximately 19-fold increase in the histone H1 phosphotransferase activity of p34cdc2. Only a single peak of histone H1 kinase activation was observed, which implies that unlike meiosis in amphibians and echinoderms, p34cdc2 is functional only during one of the meiotic divisions in S. pombe, presumably meiosis II. Stimulation of the kinase activity of p34cdc2 was associated with its tyrosyl dephosphorylation. This is analogous to mitotic M phase and suggests parallels in the mechanism of activation of p34cdc2 during mitosis and one of the meiotic divisions in S. pombe.


Transcription factor activity during cellular aging of human diploid fibroblasts. Human diploid fibroblasts display a limited proliferative life span in vitro, which is directly correlated to the age of the donor from which the cells were explanted. In an effort to identify molecular events that may underlie the loss of proliferative potential in aging fibroblasts, we have determined, at the protein level, the abundance of several cell-cycle-regulated proteins and the activity of the two major members of the activator protein-1 (AP-1) DNA binding complex. We find that cyclin A and p34cdc2 expression is decreased by two- to four-fold in old fibroblasts, but that Fos expression and binding activity are reduced by as much as 95% in old, as opposed to young cells, despite equivalent amounts of p105Rb and Jun proteins being expressed. We have further determined that the composition of the protein complex which binds a consensus (-TGACTCA-) AP-1 site changes dramatically during in vitro aging. Since we have shown previously that AP-1 activity is required for progression through the cell cycle, we propose that the quantitative and qualitative changes seen in AP-1 may play a direct role in the gradual loss of proliferative ability seen as cells approach senescence.


Factors affecting cellular mosaicism in the expression of a lacZ transgene in two-cell stage mouse embryos. In the present study, we have analysed the expression pattern of a lacZ transgene (CMZ12) in preimplantation stage mouse embryos. The transgene is expressed at the two-cell stage, where it shows cellular mosaicism due to variable expressivity. The variable gene expression indicates a partial penetrance of the transgene. The extent of variation in expression is influenced by the genetic background of the oocyte. DBA/2 and CFLP genetic backgrounds promote high expression of the transgene, while Balb/c, C57BL/6, DDK, and F1 (C57BL/6 x CBA) genetic backgrounds give none or very little lacZ activity. In vitro culture of one-cell embryos to the two-cell stage induces the expression of lacZ in all strain backgrounds tested. The variation in CMZ12 expression is a transient phenomenon and does not affect later stage activity of the transgene. Nuclear transfer experiments and DNA methylation analysis suggests that a heritable modification of the transgene locus has not occurred.


MSA-35: a protein identified by human autoantibodies that colocalizes with microtubules. We have identified a putative 35-kilodalton protein that colocalizes with microtubules and displays a unique spatial and temporal distribution during the cell cycle of HeLa cells. This protein has been given the designation MSA-35. MSA-35 first appears in association with microtubules and centrosomes of interphase cells exhibiting centrosome separation as a prelude to cell division. This protein is found in conjunction with kinetochore microtubules throughout their appearance. MSA-35 transiently associates with interpolar microtubules following anaphase and the pattern of MSA-35 reactivity in telophase cells suggests that there are at least seven domains within the intercellular bridge. The distribution of MSA-35 during and following recovery from mitotic arrest with nocodazole suggest that it is also present at low levels in interphase cells, can associate with interphase centrosomes, and colocalizes with nascent microtubules. The complex spatial and temporal distribution of MSA-35 indicates that it may be necessary for a series of events in the mitotic process such as the bundling of microtubules.


Quantitative analysis of regions of adenovirus E1A products involved in interactions with cellular proteins. Human adenovirus E1A proteins and oncogene products of several other DNA tumour viruses derive much of their oncogenic potential from interactions with cellular polypeptides. E1A proteins form complexes with p105Rb and a related p107 polypeptide, and with at least three other proteins (p60cycA, p130, and p300); all may be required for cell transformation. Using a series of E1A deletion mutants, we have carried out a quantitative analysis of the binding patterns of cellular proteins to E1A products. Binding of most of the proteins was affected at least partially by mutations within the amino terminal 25 residues, amino acids 36-69 within conserved region 1 (CR1), and residues 121-138 in conserved region 2 (CR2). However, the specific binding characteristics of each protein varied considerably. p300 was the only species for which binding was totally eliminated by deletions at the amino terminus. Removal of regions within CR1 eliminated binding of all species except p107 and p60cycA. Deletion of portions of CR2 reduced or eliminated binding of all proteins except p300. Thus, whereas cellular polypeptides generally were found to interact with the same three regions of E1A proteins, specific interactions varied considerably.


Functional analysis of the promoter of a sea urchin metallothionein gene. The 5'-flanking region of the metallothionein (MT) gene LpMT1 of the sea urchin Lytechinus pictus includes three copies of a conserved sequence that includes the metal-responsive element (MRE) consensus core sequence required for heavy metal induction of other MT genes, a GC box, a G box of a putative basal level enhancer element which includes another MRE core element, and a poly(C) tract. A fragment of LpMT1 DNA from nucleotides +31 to -309 fused to a chloramphenicol acetyltransferase reporter gene was inducible with cadmium after injection into L. pictus embryos. This induced activity was greatly reduced in a deletion mutant which retained only 195 base pairs of 5'-flanking sequence, including the proximal pair of MREs and the G box, but excluding the poly(C) tract, GC box, and distal MRE. A potent human hMT-IIA gene promoter is marginally functional in L. pictus embryos. In contrast, the LpMT1 promoter is active in HeLa cells and in embryos of the sea urchin Strongylocentrotus purpuratus. The hMT-IIA gene may lack a cis-acting sequence element required for expression of MT genes in L. pictus embryos. The LpMT1 promoter is a powerful, inducible, promiscuous promoter useful for driving the expression of heterologous genes in sea urchin embryos.


The distribution of centrosomes in migrating endothelial cells during wound healing in situ. We have examined the distribution of centrioles in rabbit thoracic aortic endothelial cells induced to migrate by wounding the endothelium in situ. Following denudation of the endothelium from a segment of the aorta with a balloon catheter, a wound edge was created from which endothelial cells began to migrate onto the denuded surface. In this in situ model of cell migration, the position of centrioles was determined in cells along the wound edge by immunofluorescence and antibodies which specifically label these cell organelles, and then they were classified in relation to the nucleus and the direction of cell migration as being oriented toward the wound, in the center, or away from wound. At time 0, as in normal unwounded adult rabbit aorta, no preferential orientation of centrioles was evident. Within 12 h after wounding, the centrioles in about 53% of endothelial cells near the wound edge were oriented toward the wound, while in less than 20% of the cells they were oriented away from wound. At 24 h, in cells up to 800 microns from the wound edge, centrioles in only about 10% of the endothelial cells were oriented away from wound, while in about 52% of cells they were found in the center and in 38% of the cells they remained oriented toward the wound. At 48 h, up to 2000 microns from the wound edge, the majority of endothelial cells had their centrioles in the center, possibly as a result of an increase in mitotic index as cells replicate to reestablish an intact endothelium.(ABSTRACT TRUNCATED AT 250 WORDS)


Cytoskeletal organization following cannabinoid treatment in undifferentiated and differentiated PC12 cells. Confocal microscopy in association with three-dimensional reconstruction was used to examine the changes in the microtubules and microfilaments following cannabinoid treatment of PC12 cells. Microtubules and microfilaments were disrupted in a dose-dependent manner following treatment with 10-30 microM delta 9-tetrahydrocannabinol (THC). A disruption of microtubules and microfilaments was observed following treatment with 30 microM cannabidiol and cannabinol. The amount of microtubules and microfilaments was reduced in a dose-dependent manner following treatment with 10 and 20 microM THC. Cannabidiol and cannabinol reduced the amount of microtubules and microfilaments; however, the reduction was less than that observed with THC treatment. Following the addition of nerve growth factor, differentiated PC12 cells were generally more sensitive to cannabinoid treatments than undifferentiated cells. The possible mechanisms that may account for the changes in microtubules and microfilaments following cannabinoid treatment are discussed.


Binding of pp60v-src to membranes: evidence for multiple membrane interactions. Membrane association of pp60v-src, the myristylated transforming protein of Rous sarcoma virus, has been shown to be a receptor-mediated process, which is inhibited by myristylated src peptides containing the N-terminal 11 amino acids of the v-src sequence (MGYsrc). By cross-linking radiolabelled MGYsrc peptide to fibroblast membranes, a 32-kilodalton membrane protein was identified as a candidate src receptor. To elucidate the potential role of p32 in binding pp60v-src, we studied the relationship between binding of MGYsrc peptide and pp60v-src polypeptide to cellular membranes. The subcellular membrane distribution of p32 was distinct from that of pp60v-src in transformed cells. Moreover, under certain defined in vitro conditions, it was possible to inhibit peptide cross-linking to p32 without significantly affecting pp60v-src membrane binding. However, when internal sequences were removed from pp60v-src, the binding characteristics of the src deletion polypeptide and MGYsrc peptide became identical. These data indicate that the presence of internal membrane binding domains influences the interaction of myristylated N-terminal src sequences with p32, and suggest that accessory binding factors might be involved in establishing stable contact between pp60v-src and the membrane phospholipid bilayer.


The effects of expression of an activated rasG mutation on the differentiation of Dictyostelium. Dictyostelium discoideum contains two ras genes, rasG and rasD, that are expressed during growth and differentiation, respectively. It was shown previously that Dictyostelium transformants expressing an activated rasD gene (a mutation producing a change in amino acid 12 from glycine to threonine) developed abnormally. When developed on filters these transformants formed multitipped aggregates, which did not go on to produce final fruiting bodies, but in a submerged culture assay on a plastic surface they either formed small aggregates or did not aggregate. In this study we transformed cells with the rasG gene, mutated to change amino acid 12 from glycine to threonine. The resulting transformants developed normally on filters, but aggregation under other conditions was impaired. In particular, in submerged culture on a plastic surface they either produced very small aggregates or did not aggregate, one of the phenotypes exhibited by the activated rasD transformants. Molecular analysis of the transformants revealed the presence of high copy numbers of the mutated rasG gene, but the level of expression of the mutant gene never exceeded the level of expression of the endogenous gene. These results indicate a powerful dominant effect of a relatively small amount of the activated RasG protein in Dictyostelium.


Zygote giant cell differentiation in Dictyostelium discoideum: biochemical markers of specific stages of sexual development. Sexual development in Dictyostelium discoideum has many unique features making it an attractive eukaryotic model system for the study of biomembrane fusion and intercellular communication. The work presented here provides primary biochemical evidence for two distinct phases during early sexual development that appear to be defined by calcium-dependent gamete cell fusion. In addition, we introduce a novel procedure for the enrichment of zygote giant cells and use this method to define certain wheat-germ agglutinin binding glycoproteins which are specifically located in zygote giant cells and others which are markers for surrounding amoebae in the second phase of development. In addition, a G protein which is present in high amounts early in development is unique to giant cells in the second phase, suggesting a role in phagocytosis. Finally, alkaline phosphatase activity was found to mark the first phase of sexual development, suggesting a role in cell fusion. This contrasts with the patterns of alpha-mannosidase and beta-glucosidase activity that increase late in the second developmental phase, where they likely function in endocyte digestion during the cytophagic period. The developmental significance of these findings is discussed.


The fate of female donor blastodermal cells in male chimeric chickens. In previous experiments in our laboratories, chickens that are chimeric in their gamete, melanocyte, and blood cell populations have been produced by injection of dispersed stage X blastodermal donor cells into the subgerminal cavity of stage X recipient embryos. In some experiments, donor cells were transfected with reporter gene constructs prior to injection as a preliminary step in the production of transgenic birds. Chimerism was assessed by test mating, observation of plumage, and DNA fingerprinting. Methods were sought that would provide a relatively rapid analysis of the spatial distribution of descendants of donor cells in chimeras to assess the efficacy of various methods of chimera construction. To date, the sex of donor and recipient embryos was not known and, therefore, numerous mixed sex chimeras must have been constructed by chance, since donor cells were usually collected from several embryos rather than from individual embryos. The presence of female-derived cells was determined by in situ hybridization using a W-chromosome-specific DNA probe, using smears of washed erythrocytes from 16 phenotypically male chimeric chickens ranging in age from 4 days to 42 months posthatching. The proportion of female cells detected in the erythrocyte samples was zero (eight samples) or very low (0.020-0.083%), although 1% of the erythrocytes from a phenotypically male chick that was killed 4 days after hatch were female-derived. The low proportions of female-derived cells were surprising, considering that most of these chimeras had been produced by the injection of cells pooled from several donor embryos and most recipients had been exposed to gamma irradiation prior to injection, thus dramatically enhancing the level of incorporation of donor cells into the resulting chimeras. By contrast, 0-100% of the erythrocytes were female-derived in blood samples taken at 10 days of incubation from the chorioallantois of seven phenotypically normal male embryos that resulted from the injection of blastodermal cells pooled from five embryos into irradiated recipient embryos. Approximately 70% of the erythrocytes in a blood sample from a phenotypically normal female chimeric embryo were female-derived, and 100% of the erythrocytes examined from an intersex embryo bearing a right testis and a left ovary were female-derived. These results indicate that female-derived cells can contribute to the formation of erythropoietic tissue during the early development of what will become a phenotypically male chimeric embryo. It would appear, therefore, that female-derived cells are blocked in development or destroyed, or certain male-female combinations of cells may be lethal prior to hatching.(ABSTRACT TRUNCATED AT 400 WORDS)


Mutagenesis of Ste18, a putative G gamma subunit in the Saccharomyces cerevisiae pheromone response pathway. The yeast STE18 gene product has sequence and functional similarity to the gamma subunits of G proteins. The cloned STE18 gene was subjected to a saturation mutagenesis using doped oligonucleotides. The populations of mutant genes were screened for two classes of STE18 mutations, those that allowed for increased mating of a strain containing a defective STE4 gene (compensators) and those that inhibited mating even in the presence of a functional STE18 gene (dominant negatives). Three amino acid substitutions that enhanced mating in a specific STE4 (G beta) point mutant background were identified. These compensatory mutations were allele specific and had no detectable phenotype of their own; they may define residues that mediate an association between the G beta and G gamma subunits or in the association of the G beta gamma subunit with other components of the signalling pathway. Several dominant negative mutations were also identified, including two C terminal truncations. These mutant proteins were unable to function in signal transduction by themselves, but they prevented signal transduction mediated by pheromone, as well as the constitutive signalling which is present in cells defective in the GPA1 (G alpha) gene. These mutant proteins may sequester G beta or some other component of the signalling machinery in a nonfunctional complex.


Ras modulates commitment and maturation of 10T1/2 fibroblasts to adipocytes. The positive association of the ras oncogene with human cancer and the recognition that malignancy may, in part, represent the imbalance between cell proliferation and differentiation have generated intense interest in the potential role of ras in cell differentiation. We investigated this possibility utilizing as a model system the differentiation of the mesenchymal cell line C3H 10T1/2 (10T1/2) to adipocytes, and a series of transfectants of 10T1/2 cells in which the level of the ras gene product (p21ras; Ras) can be effectively up- or down-modulated. In agreement with previous reports, we found that 10T1/2 cultures, propagated in the resting state for several weeks, spontaneously convert to fat cells at a very low frequency. Downmodulation of endogenous p21ras levels, as a consequence of expression of antisense ras, markedly increased the rapidity and frequency of adipose conversion (6- to 10-fold), which was equivalent in magnitude to that effected by the potent differentiating agent 5-azacytidine. Conversely, overexpression of ras completely inhibited cell differentiation. In addition, adipocytes derived from antisense-ras expressing lines were characterized by a decrease in hormone responsiveness, as well as an apparent deficiency in attaining the terminally differentiated state. These findings suggest that Ras may be a negative regulator of the decision-making step of fibroblast differentiation to adipocytes. In addition, Ras may play an essential positive role in the transduction of hormonal signals necessary for full adipocyte maturation during later progression along the differentiation pathway.


Resistance of ADP-ribosylated histones and HMG proteins to proteases. Calf thymus histones (individually isolated or mixtures) and high mobility group proteins were ADP-ribosylated in vitro using [32P]NAD+ and immobilized purified poly(ADP-ribose) polymerase. The modified histones were then subjected to V8 protease or alpha-chymotrypsin digestion and the resulting peptides were separated by electrophoresis on acetic acid-urea-Triton gels. It was found that in vitro ADP-ribosylated histones were much more resistant to proteases than unmodified histones. A similar approach was applied to histones modified by the endogenous poly(ADP-ribose) polymerase in permeabilized NS-1 mouse myeloma cells in culture. In this case, the proteases could not discriminate between modified and unmodified histones and putative mono(ADP-ribosyl)ated peptides appeared in a digestion frame corresponding to that of bulk peptides. These differences are most probably due to the specificity or number of ADP-ribose groups added to the histones by the endogenous or exogenous poly(ADP-ribose) polymerase. Thus, depending on the size of poly(ADP-ribose) attached to nuclear proteins, these modified proteins might display different degrees of resistance to proteolysis.


The origin of the cancer cell: oncogeny reverses phylogeny. The formulation in 1874 of the biogenetic law by Ernst Haeckel as "ontogeny recapitulates phylogeny" emphasized the structural similarities detected in metazoans between their developmental and ancestral forms. More recently, many workers have independently commented on the similarities observed between the behaviours displayed by dedifferentiated cancer cells and their embryonic precursors. This review will explore a possible linkage between these disparate observations and will suggest that cancer cells behave in ways that are reminiscent of primitive eukaryotic cells. In particular, we suggest that the acquisition of a multicellular level of organization during early metazoan evolution required a critical and difficult change in growth strategy as germ line and somatic cells became distinct. Whereas unicellular free living eukaryotes follow a simple strategy of rapid division as long as conditions permit, the elaboration of powerful growth inhibitory pathways must have been necessary in primitive multicellular organisms to enable some but not all sister cells to stop dividing, even under conditions of nutrient abundance. This limitation on cellular growth would than have permitted the appearance of tissues and organs with differentiated characteristics, ultimately enabling the enhanced survival of the meiotic lineage. Cancer cells might therefore be considered to represent, with their loss of tumor suppressor inhibitory activity and elevation of oncogene stimulatory activity, a reversion to a more primitive evolutionary state capable of indeterminate growth at the expense of the host. By this analogy, the growth phenotypes displayed by cancer cells, embryonic cells, and free-living eukaryotes are fundamentally similar.


Towards an understanding of microtubule function and cell organization: an overview. Microtubules exhibit dynamic instability, converting abruptly between assembly and disassembly with continued growth dependent on the presence of a tubulin-GTP cap at the plus end of the organelle. Tubulin, the main structural protein of microtubules, is a heterodimer composed of related polypeptides termed alpha-tubulin and beta-tubulin. Most eukaryotic cells possess several isoforms of the alpha- and beta-tubulins, as well as gamma-tubulin, an isoform restricted to the centrosome. The isoforms of tubulin arise either as the products of different genes or by posttranslational processes and their synthesis is subject to regulation. Tubulin isoforms coassemble with one another and isoform composition does not appear to determine whether a microtubule is able to carry out one particular activity or another. However, the posttranslational modification of polymerized tubulin may provide chemical signals which designate microtubules for a certain function. Microtubules interact with proteins called microtubule-associated proteins (MAPs) and they can be divided into two groups. The structural MAPs stimulate tubulin assembly, enhance microtubule stability, and influence the spatial distribution of microtubules within cells. The dynamic MAPs take advantage of microtubule polarity and organization to vectorially translocate cellular components. The interactions between microtubules and MAPs contribute to the structural-functional integration that characterizes eukaryotic cells.


Intermediate filament proteins: many unanswered questions, few unquestioned answers. Major constituents of the cytoskeleton and the nuclear matrix, cytoplasmic intermediate filament subunits and nuclear lamins belong to a multigene family of proteins whose function is poorly understood. It has now become a general contention that important clues to the physiological roles of these proteins may reside in their developmental and tissue-specific expression patterns, as well as their cellular organization. The present review brings into focus experimental strategies that have been developed, over the past few years, to gain insights into the cellular mechanisms regulating the molecular polymorphism and supramolecular assembly of intermediate filaments. In this context new concepts are discussed that may be pivotal for the orientation of future studies on intermediate filament proteins.


Nuclear and chromatin composition of mammalian gametes and early embryos. Changes in nuclear structure and chromatin composition regulate gene activity in many cell types and could play a similar role during early mammalian embryogenesis. Oocytes of the mouse contain the three major lamin species present in somatic cells, although lamin A synthesized by oocytes has a higher molecular mass than the somatic species. Oocyte chromatin contains core histones similar to those of somatic cells, as well as elements that are immunologically related to protamines. In contrast, somatic-type histone H1 is not present. DNA topoisomerase II has not yet been identified in mammalian oocytes, but is abundant in frog oocytes. In contrast to oocytes, sperm do not contain a typical nuclear lamina. DNA topoisomerase II is detectable until late spermiogenesis. Although the DNA of sperm is associated mainly with protamines, some histone may be retained. There is also evidence that the arrangement of the DNA in the nucleus is nonrandom. These results demonstrate differences in nuclear and chromatin composition between oocytes and sperm. After fertilization, the nuclei of cleavage-stage blastomeres undergo programmed modifications. Lamin B is synthesized, whereas lamin A is not. In addition, a set of nuclear proteins is transiently synthesized in mice at the two-cell stage. Changes in embryonic chromatin composition also occur. The relative abundance of transcripts from different core histone genes differs between mouse oocytes and blastocysts. Furthermore, somatic histone H1 becomes detectable beginning at the mid-four-cell stage. As well, during early cleavage stages, expression of plasmid-borne genes becomes dependent on enhancers. Thus, developmentally regulated changes in nuclear and chromatin composition occur during early mammalian embryogenesis, and these may be important for the initiation and regulation of embryonic gene activity.


Mechanisms of placental invasion of the uterus and their control. Trophoblast cells of the placenta in many species have acquired mechanisms to invade the uterus, inclusive of its blood vessels, to establish efficient fetomaternal exchange of molecules. This invasion is strictly controlled both spatially and temporally and, in humans, usually continues until midgestation. Key mechanisms underlying various steps in trophoblast invasion are: (i) the attachment to the basement membrane, most likely by binding to laminin; (ii) the detachment from the basement membrane matrix, a process requiring the presence of complex-type oligosaccharides on the cell surface; and (iii) the breakdown of basement membrane components, mediated by secretion of metalloproteases (such as type IV collagenases) and serine proteases (plasminogen activator). Activation of trophoblast-derived metalloproteases appears to be plasmin dependent. Trophoblast invasiveness in situ is controlled by the microenvironment, owing to local production of anti-invasive factors by the decidual tissue of the uterus. One of these factors is TIMP (tissue inhibitor of metalloproteases), which neutralizes metalloproteases in an equimolar ratio. Another is TGF-beta (transforming growth factor-beta), which has a dual effect: it induces TIMP-1 secretion by the trophoblast and decidual cells and promotes differentiation of invasive trophoblast cells into multinucleated giant cells, which are presumably noninvasive. Thus, TGF-beta provides the key control of trophoblast invasiveness in situ. This control is lost in certain choriocarcinomas. In contrast to the response shown by the normal trophoblast, JAR and JEG-3 choriocarcinoma cell invasiveness does not seem to be inhibited by TGF-beta. In fact, in preliminary studies, JAR cells responded to TGF-beta by increased invasiveness.(ABSTRACT TRUNCATED AT 250 WORDS)


Towards understanding the control of the division cycle in animal cells. The author reviewed the historical process by which classical knowledge of cell division accumulated, to give rise to the molecular biology of the cell cycle, and discussed the perspective of this field of research. The study of the control of cell division began at the turn of the century. It was hypothesized that cell division was a physiological regulation necessary for growing cells to maintain a proper nucleocytoplasmic ratio to survive, which was later substantiated by the finding that amoeba cells could be prevented from dividing by repeated excision of the cytoplasm. However, the observation in Tetrahymena that heat-shocked cells grow exceedingly, but fail to divide, suggested that the cell required the accumulation of a labile "division protein" to initiate division. Mechanisms that control the cell cycle were studied in oocytes by nuclear transplantation and cytoplasmic transfer, and in cultured mammalian cells, protozoa, and Physarum plasmodia by cell fusion. These experiments demonstrated the existence of cytoplasmic factors that control the cell cycle. Maturation promoting factor (MPF) thus discovered in frog oocytes became known to be an ubiquitous cytoplasmic factor that causes the transition from interphase to metaphase in all organisms. The insight into the molecular control of cell growth and division was gained from yeast cell genetics. For biochemical analysis of the cell cycle control, the method to observe the cell cycle in vitro was developed using frog egg extracts. Thus, MPF was identified as a cdc2--cyclin protein complex. Its activity was found to depend on synthesis and phosphorylation of these proteins. However, recently it was found that there were cell cycle phenomena that were difficult to explain in these terms. Various other cellular factors, including nucleocytoplasmic ratio and microtubule assembly, were also found to control MPF, as well as the cell cycle. It remained open to future how these factors control MPF to alter the pattern of the cell cycle.


Regulation of the G2-mitosis transition. The cell cycle is regulated by pathways composed of a dependent series of steps, by timers, and by checkpoint controls which ensure the completion of one event before the initiation of another. This review focuses on the regulation of the initiation of mitosis, with particular emphasis on the regulation of p34cdc2 activity at this point in the cell cycle. The review draws on data from various organisms, but strongly emphasizes the genetic framework as seen in the fission yeast Schizosaccharomyces pombe and the biology and biochemistry of maturation promoting factor in frog oocytes. An attempt is made to include all known genes and proteins where a link can be made to the initiation event. The nutritional size control and its major known controlling elements, the wee1/mik1 protein kinases, and cdc25 protein tyrosine phosphatase are considered in detail along with their regulation. In addition, the checkpoint control pathways which mediate G2 delay in response to failure of DNA replication or DNA damage are examined.


[The shape of the nasopharynx in youth: statistical study]. The particular shape of the nasopharynx in the child can influence morphology and physiopathologic behaviour of the mucosa. In such a perspective, it appeared interesting to evaluate the kind and the entity of its variations by means of nuclear magnetic resonance (NMR) in 50 patients (age range: 2 months-16 years). The results show that the nasopharyngeal shape (represented either by bending radius and shape factor) varies in a statistically significant way during the considered age range, while the dimension of the pharyngeal tonsil remains constant in the studied cases. The statistically significant shape variation of the tonsil appears, so, related to its involvement in the shape variation of the whole nasopharynx.


Calcium storage in nonmuscle tissues: is the retina special? It is now well established that calreticulin is a high capacity Ca(2+)-binding protein which is a major Ca2+ storage protein of the lumen of endoplasmic reticulum membranes in a wide variety of tissues with the exception of skeletal and cardiac muscles. However, in nervous tissue, confusion exists regarding the nature of the intracellular Ca2+ stores, as the organelle responsible for Ca2+ storage has been identified as the endoplasmic reticulum by some investigators and as the specialized organelle, calciosome by others. Calreticulin, calsequestrin, and calsequestrin-like proteins have all been, on different occasions, reported to be present in calciosomes. Cerebral and cerebellar tissues, moreover, have been shown to contain somewhat different systems of Ca(2+)-buffering proteins. In the present paper we discuss evidence that the Ca2+ storage systems of the retina may prove to be more complex than those of other neuronal tissues. Biochemical and immunocytochemical evidence indicates the presence of either an isoform of calreticulin or another protein that is antigenically similar to calreticulin, but of slightly higher molecular weight, in the endoplasmic reticulum of both neurons and Müller glia from rabbit neural retina. However, as retinal neurons express Purkinje cell markers, one may expect to observe the presence of calsequestrin in these cells as well. Secondly, antibodies against the onchocercal RAL-1 antigen recognize a protein sharing 62-65% amino acid sequence identity with calreticulin. The anti-RAL-1 antibodies show specificity for the retina. Whether or not the RAL-1 antigen is an active part of the Ca2+ storage systems of the retina remains to be verified.(ABSTRACT TRUNCATED AT 250 WORDS)


G1 cyclins regulate proliferation of the budding yeast Saccharomyces cerevisiae. The eukaryotic cell cycle is regulated at two points, the G1-S and G2-M boundaries. The molecular basis for these regulatory activities has recently been elucidated, in large part by the use of molecular and genetic analyses using unicellular yeast. The molecular characterization of cell-cycle regulation has revealed striking functional conservation among evolutionarily diverse cell types. For many eukaryotic cells, regulation of cell proliferation occurs primarily in the G1 interval. The G1 regulatory step, termed START, requires the activation of a highly conserved p34 protein kinase by association with a functionally redundant family of proteins, the G1 cyclins. Here we review studies using the genetically tractable budding yeast Saccharomyces cerevisiae, which have provided insight into the role of G1 cyclins in the regulation of START.


Regulation of gene expression in oncogenically transformed cells. Several genes expressed in response to growth factors are also regulated aberrantly in oncogenically transformed cells. The constitutive expression of genes encoding extracellular proteases, transcription factors, and cytokines is often correlated with cell transformation. In several instances, the uncontrolled expression of these genes is the result of transcriptional activation. Therefore, much attention has been devoted to the study of promoter function in transformed cells. We now review the results of recent investigations on transformation-dependent gene expression. The activation of several transcription factors in oncogenically-transformed cells is described. Results regarding the regulation of promoters through PRD II/kappa B are presented for cells transformed by a variety of oncogenes. Finally, we discuss the significance of transcription factor activation in the process of cell transformation.


[Study of the interaction between plasma proteoglycans and LDL by means of fluorescence spectroscopy]. The relevance of the interaction between LDL and PGs in the development of atherosclerotic processes is well known. However, the exact nature of the interaction and the consequent structural and/or conformational modifications of the lipoprotein remain to be clarified. It has been demonstrated that after this interaction the LDL particle is not recognized by specific cellular receptors and enters the scavenger pathway operating in different cell types. These effects have been shown by using aortic PGs, but PGs are also present in the plasma compartment and may interact constantly with LDL, taking part in the regulation of lipid metabolism. In order to assess the capability of plasma PGs to induce LDL modifications, we investigated their interactions by studying the changes in the organizational parameters of LDL by fluorescence spectroscopy. Plasma PGs were isolated by DEAE Sephacel ion exchange chromatography and Sephacryl S300 gel filtration in two different families: a low-charge PG and a high-charge PG. Human LDL was prepared from plasma of normolipemic donors by ultracentrifugal flotation between 1.025-1.045 g/ml. Steady-state anisotropy measures were obtained by analyzing the rotational diffusion rate of DPH after incubation of LDL with plasma PGs in a physiological ratio. In our experimental conditions, LDL incubation with plasma low-charge PG did not modify DPH fluorescence anisotropy, whereas LDL treatment with highly charged PGs induced a marked decrease of this parameter, suggesting a significant effect on LDL microviscosity. The data show that both the charge and the GAG composition of PGs appear to be critical factors in LDL-PG interaction.(ABSTRACT TRUNCATED AT 250 WORDS)


Multiple antigens recognized by anti-c-myc antibodies in human cells and Xenopus oocytes. We have investigated the localization, solubility, serum regulation, and phosphorylation of MYC antigens from Colo 320 cells, a human transformed cell line with an amplified c-myc gene, and from Xenopus oocytes, which express high levels of c-myc mRNA. Although MYC proteins are often reported to range from 60 to 68 kilodaltons, our panel of anti-MYC monoclonal antibodies recognized a number of higher and lower molecular mass antigens, in addition to proteins within this range. Based upon various criteria, including cross-recognition by several anti-MYC antibodies, we suggest that some of these antigens are bona fide MYC family proteins. Our results, as well as those of others reported previously, suggest that several MYC antigens may be simultaneously present in cells. The apparent diversity among members of the MYC family of antigens raises the possibility of multiple cellular functions and regulatory roles for these proteins.


Parietal neurons encoding visual space in a head-frame of reference. Neurons of the visual system are known to have receptive fields organized in retinotopic coordinates. We wanted to test whether visual neurons existed whose receptive fields were organized in spatial coordinates. Extracellular recordings from single cells were carried out in one area of the posterior parietal cortex (area V6) of a behaving macaque monkey. Among a great majority of retinotopically organized visual cells, neurons whose visual receptive field did not shift with gaze were also found. These cells responded to the visual stimulation of the same spatial position independently of the animal's direction of gaze, that is, their receptive field was anchored to an absolute spatial location. We suggest that these neurons directly encode visual space and are involved in programming visually-guided motor actions in space.


Presence of antisperm antibodies in the serum of Sardinian adult people. II. Study on a selected infertile population in north Sardinia. Among many possible causes of infertility an immunological factor must be considered, in particular the presence of antibodies to spermatozoa in the serum. In previous researches we confirmed the presence of antisperm antibodies in the serum of healthy men of North Sardinia in percentage equal to 2.4% of the considered population. The aim of the present study is to evaluate the correlation between the presence of antisperm antibodies and infertility. The presence of antispermatozoal antibodies has been evaluated in the serum of 124 men (aged 25-50) affected by unexplained infertility utilizing the indirect immunofluorescence and ELISA methods. The presence of antisperm antibodies has been found equal to 19.3% of the tested sera. These results show that the incidence of antisperm antibodies is higher in the serum of male partners from infertile couples than in healthy subjects (P < 0.01), also in a relatively segregated and pure population like the one studied.


Computerized topographical analysis of functionally homogeneous neuronal sets. A computer-assisted analysis of the spatial distribution of neurons having homogeneous characteristics is described in this paper. The camera lucida drawings of sections of a brain nucleus and the points representing the neurons labeled on the basis of a specific behavior of discharge rates were digitized on a personal computer Amiga 2000 or IBM compatible. Our software provided: a) the computerized, stereotaxically oriented reconstruction of the stored sections and of the plotted neurons; b) the identification within each section of the mass center (MC) of the units sharing a given behavior and of the area where the density of such neurons was maximal (MDA). The routine was tested on the spatial distribution of neuronal responses to serotonin in the lateral vestibular nucleus.


[Microsurgery and laser surgery in conservative operations on the ovary. Experimental research]. In this study we compared two different conservative surgery techniques performed on 12 ovaries of female rabbits: microsurgery and CO2 Laser surgery. After the surgical procedure all the animals were investigated by a Laparotomy to evaluate the post-operative adhesion formation. Histological examinations were performed on 6 ovaries, to evaluate the possible damage to the ovarian parenchyma. We did not find significant differences between the two methods employed, particularly for the adherence formation and the parenchymal thermic damages: no post-operative adhesions were detected respectively in 3 ovaries operated on by microsurgery and 5 by laser surgery; slight adhesions were present in 2 ovaries treated with microsurgery and in 3 with laser surgery; 3 ovaries treated with microsurgery and 2 with laser surgery showed moderate adhesions. Only 2 ovaries treated with microsurgery presented severe adhesions.


Multi-system approach to study mutagenesis induced by chemical carcinogens. To understand molecular mechanisms of the mutation fixation process induced by a mutagen and carcinogen, a multi-system approach is suggested to reduce the probability that the results are biased by the assay used. In this light we described our different approaches to answer basic questions on the mutagenesis induced by the chemical carcinogen 4-Nitroquinoline-1-oxide. We determined mutations at the molecular level in three experimental systems: a) in prokaryotes (ss M13mp19 lacZ'/E. coli F'lacZ delta M15); b) in eukaryotes (i) ss and ds pZ189 supF/CV1-P/E.coli lacZam and (ii) HPRT in CHO cells with different repair capacity. We think this type of approach can be used to study the genetic effects of new cancer drugs for which the molecular mechanisms of action at the molecular level are still not well understood. We think to apply the know-how to study mutational spectra in tumor derived tumor suppressor genes.


[Critical ratio: effects of contralateral masking on normal and pathological ears]. In order to evaluate the central interferences on the auditory efficiency, the effect of contralateral masking noise on Critical Ratio (CR) has been studied in 14 normal ears (control group), in 12 ears with cochlear disorders and in 6 with retrocochlear lesion (acoustic neuromas); in all cases the impairment was unilateral, while the other ear was normal. CR values were calculated for 1 KHz pulsed tones (duration 500 ms, rise/fall 25 ms, duty cycle of 50%); the masking noise was a wide band (90-20,000 Hz) delivered at 40 dB SL. The results have demonstrated that in the presence of contralateral masking noise, CRs don't modify both in the normal ears and in those with retrocochlear disorder, whereas they increase, almost always, in ears with cochlear deafness. These results demonstrate that the involvement of central auditory pathways, because of contralateral noise, makes the auditory efficiency worse, only in ears with cochlear dysfunctions; this behaviour seems to confirm the peripheral origin of CR.


Diagnosis and treatment of asthma in children: usefulness of a review of medical records. In order to tackle the problems of underdiagnosis and undertreatment of asthma in childhood general practitioners need to be aware of which children in their practices have or might have asthma. In an effort to identify a cohort of asthmatic or potentially asthmatic children a trained audit facilitator studied all the medical records of children aged between one year and 15 years who were registered with 12 Tayside general practices. From a total of 10,685 medical records the frequency of 'key items' sometimes associated with asthma were as follows: one or more episodes of bronchospasm or wheeze 23.7% of children, persistent cough 23.2%, treatment with anti-asthma therapy in the past 20.0%, exercise induced cough or wheeze 5.2% and history of 'wheezy bronchitis' 4.6%. However, in only 896 children (8.4%) had a formal diagnosis of asthma been made. Of all the children, 5.4% had received a prescription for anti-asthma medication within the past three months. Only 1.2% were taking an inhaled corticosteroid and 1.0% sodium cromoglycate, but many more were taking inhaled bronchodilators (3.1%) and oral bronchodilators (1.7%). The findings suggest that a systematic review of medical records by a trained facilitator can identify those children who could benefit from clinical review. Practices who wish to know which of their children have or might have asthma should consider using medical record review to search for key items associated with asthma.


Schoolteachers' perceptions and knowledge of asthma in primary schoolchildren. Schoolteachers are known to be concerned about asthma in their pupils but their opinions about the best method of addressing this concern have not previously been investigated. Eleven headteachers and 76 class teachers from 11 primary schools in the Southampton area--eight from the state sector and three private day schools--completed questionnaires. These inquired about the organization of care for asthmatic pupils, the teachers' knowledge, concerns and training regarding the disease, and their perceptions of the need for and source of further education for teachers about asthma. All 10 National Health Service school health services in the Wessex region and 16 teacher training colleges responded to questions about their provision of such education for teachers. The results demonstrated that asthma care in the 11 schools was generally disorganized, with the responsibility for keeping and administering inhalers falling on the school secretary in seven of the schools. All but two of the 11 headteachers and 89% of the class teachers were concerned about asthma, primarily in managing emergencies. Levels of knowledge were low, particularly regarding exercise, and education about asthma was almost non-existent. The great majority of class teachers (86%) wanted more information about asthma and most (67%) looked to the school health services to provide this. None of the school health services in Wessex and only one teacher training college had any planned education about asthma for teachers. Clearly, schools need to receive more information about asthma, both to enable them to cope more ably with their asthmatic pupils and to allay the anxieties of teachers.(ABSTRACT TRUNCATED AT 250 WORDS)


How effective is systematic care of diabetic patients? A study in one general practice. A method of systematic diabetic care compatible with personal lists, the 'diabetic day', was introduced into a seven partner inner city general practice. The effect on glycosylated haemoglobin levels and the recording of six process measures (fundoscopy, visual acuity, weight, blood glucose levels, glycosylated haemoglobin levels and blood pressure) was assessed. Of the 111 known registered diabetic patients, 64 entered the diabetic day and fulfilled the eligibility criteria. General practice records were analysed retrospectively over a period of four years--the two years before entry into the diabetic day were compared with the subsequent two years. Mean glycosylated haemoglobin levels fell from 10.52% in the year before entry to the diabetic day to 9.71% in the second year after entry (P < 0.01, 95% confidence intervals 0.19 to 1.39). There was a significant increase in all process measures recorded in the general practice notes after entry into the diabetic day. The introduction of systematic care for diabetic patients led to an improvement in recorded process measures and a reduction in patients' glycosylated haemoglobin levels in a general practice which had made previous efforts to improve diabetic care and was already well staffed, organized and motivated.


What do medical students seek to learn from general practice? A study of personal learning objectives. With the aim of stimulating learning which is more self directed, fourth year medical students in Liverpool are encouraged to set personal learning objectives for the general practice attachment. On average, a student defines seven objectives for the three week attachment. A classification of objectives derived from the 1989 cohort of students is presented and the objectives could be seen as focusing on the practice population and its health problems, the role of the general practitioner, the work of general practice, the management of general practice, general practice as a career, and general learning. The validity and reliability of the classification are considered. Along with the advantages of this approach in motivating students to learn, the findings are considered in relation to impending changes in undergraduate medical education and the future role of general practice teaching by departments and by practice based colleagues.


Practice organization before and after the new contract: a survey of general practices in Sheffield. In order to assess the effects of the new contract on practice organization, all general practices in Sheffield were surveyed just before the new contract came into effect in April 1990, and again one year later. Of the 120 practices, 57% responded in 1990 and 61% in 1991, with 47% responding in both years. There were significant increases in the mean number of clinics and employed staff for the practices responding to both questionnaires and in the proportion of these practices which had a computer. These changes represent a response to the incentives and stated aims of the new contract.


Preventable deaths: 16 year study of consecutive deaths in a village in Israel. The aims of this study were to examine mortality in one village in Israel and to determine which deaths could have been prevented by identifying those which were associated with avoidable factors or were caused by conditions which would have been amenable to preventive measures. The medical records of all 171 patients (91 males and 80 females) who died in the geographically defined population of 1800 during the 16 year period 1974-89 were reviewed. The mortality rate, adjusted for age, in the second eight year period (1982-89), was significantly lower for females (4.0 deaths per 1000 females per year) than for males (8.5) (P < 0.01). Downward trends in the birth rate, stillbirth rate and perinatal mortality rate were found, while there was an upward trend in both mean and median age at death. Of the 171 deaths, 36 (21%) were classified as being associated with a total of 44 factors which could be perceived as being avoidable. Twenty five of the avoidable factors were patient related and of these 17 were smoking (more than 20 cigarettes a day in patients aged less than 70 years who died of a smoking related disease). These findings confirm the need for continuous health education for patients. An audit of mortality in general practice is valuable and may contribute towards the prevention of some deaths.


General practitioners and emergency treatment for patients with suspected myocardial infarction: last chance for excellence? Pre-hospital coronary care usually consists of a medically staffed coronary care ambulance going into the community from a hospital base, as pioneered in Northern Ireland. In today's medicopolitical and economic climate, this model is not viable in mainland United Kingdom. Current proposals seem to favour a 'scoop and run' policy for heart attack victims, that utilizes the ambulance service but bypasses the general practitioner. Since the majority of telephone calls from people with suspected myocardial infarction are directed to general practitioners, a preferable alternative would be a 'stay and stabilize' strategy that uses the existing referral pattern and builds on general practitioners' medical education and skills. The role of the general practitioner in the management of patients with suspected myocardial infarction is discussed.


[The fracture risk in the elderly subject: the determining factors]. Recent advancements in the field of bone mass measurements allow us a precise determination of bone mineral density values in various skeletal sites. At present, their determination gives the most valuable information on individual fracture risk. However, we sometimes to witness fragility fractures in presence of regular values of bone mineral density which suggest a satisfactory status of skeletal mineralization. These cases underline that the mechanical incompetence of the skeleton is not simply the result of a decrease in bone mineral density and that other factors are responsible for the increase of fracture incidence in the elderly. In this paper, several factors potentially involved in the occurrence of fragility fractures in elderly people have been considered on the basis of the most recent data of the current literature in this field. These factors, subdivided in bone tissue extrinsic and bone tissue intrinsic factors, include: muscle synergisms, reflex reactions and sensory status, soft tissue features, bone tissue features, structural properties and loading modalities in relation to structural orientation. Awareness and analysis on these factors are important for better management of preventive as well as therapeutic measures for fragility fractures in elderly.


[Treatment experience with chronic spinal pain in involutional osteoporosis]. The back pain syndrome which accompanies involutional osteoporosis presents a marked heterogeneity. Acute pain may be due to vertebral fractures, whereas chronic pain may eventually accompany established osteoporosis in which clinical and instrumental evidence are present. Back pain is the consequence of the mechanical (internal or external pressure) or chemical stimulation of pain receptors present in bone tissue, along the vessels, in cartilage, joints, disk, ligaments, and also in soft tissue and muscle (with secondary antalgic contracture). The compression of spinal nerves may contribute to the pain as well. An evident alteration of mood is usually present and represents an important element in the syndrome. This phenomenon interferes with the evolution of pain, in particular as regards its intensity. Besides scales for the evaluation of pain and inability, it is possible to check objective data by means of particular algometers (not easy to employ) or by electromyographic measurements of antalgic secondary contracture of spinal muscle. Gait examination (basography) of patients with painful hip prosthesis may provide objective evaluation regarding specific antalgic activity on bone of drugs. Usually the effective drugs for osteoporosis possess antalgic properties as well, with different mechanisms of action. Three drugs with evident activity are taken into consideration: calcitonin, ipriflavon, aminobutane-bisphosphonate (alendronate). Though each of them possesses some particular activity, the main mechanism of action is dependent on their effect on the local microenvironment, particularly at the level of bone tissue (calcium, cytokine and prostaglandin local concentration), on the modulation of osteoclast activity. In particular alendronate (intermittently administered intravenously) exerts the most evident antalgic activity. Subjective chronic back pain relief is accompanied by (secondary) reduction of antalgic contracture at vertebral muscle level. The activity of the substance against the painful hip prosthesis (documented by basographic gait recording) leads us to conclude that the substance really exerts a direct antalgic action at the level of bone tissue.


The future of osteoporosis diagnosis and therapy. Advancing the diagnosis and treatment of osteoporosis will require progress on several fronts: we must overcome the current widespread failure to fully employ the means already at our disposal for the evaluation, prevention and treatment of osteoporosis. Diagnostic testing must become more effective at characterizing the bone and mineral metabolism of the individual patient. Testing must do far more than identify candidates for treatment; it must permit monitoring of the response to therapy or progression of disease. New therapies should have well-characterized, very specific physiological effects. It is critical that they do not produce adverse effects on the bone remodeling mechanism. Gaps in our understanding of the relationships between the mechanical functions of the skeleton and the biology of bone tissue must be filled in order to provide a basis for evaluation and treatment of the skeleton as an integrated organ system.


[Therapeutic strategies in osteoporosis: whom and when to treat]. Postmenopausal bone loss can be efficiently prevented by compounds which inhibit bone resorption, but they can not induce a sustained and relevant bone gain. The initial increase in bone volume observed within the first year of treatment is due to a transient uncoupling between bone resorption and formation: the first is suddenly blocked whereas bone formation decreases slowly within several months; bone is gained until the previously resorbed cavities are refilled. This net increase in bone mass is completely lost as soon as treatment is withdrawn. Since these antiresorptive agents can only be used in order to prevent bone loss, they should be given for several years. Thus, the choice of the drug is also conditioned by its cost and long-term compliance. Estrogen replacement is very convenient, from these points of view in most patients immediately after the menopause. Afterwards, parenteral calcitonin has been, up to now, the sole available alternative with convincing evidence of effectiveness, at least in the medium term. Its cost and the scarce compliance to injections have strongly limited its extensive use. There is increasing evidence that oral bisphosphonates are very effective and therefore they might represent, in the near future, an effective and convenient alternative to estrogen replacement therapy.


[Symptomatic headaches in internal medicine: the classification, physiopathological and diagnostic aspects]. Symptomatic or secondary headache occurs when pain itself is a symptom of disease. It is well known that within the general population the percent frequency of secondary headache is lower than that of primary headache. Moreover, some forms do not seem to evidence particular clinical, diagnostic or physiopathological importance. The Authors investigate here a number of clinical aspects of secondary headache, in particular headache in vascular disease (stroke, hypertension, Horton's arteritis). Particular attention is paid to headache in brain neoplasia due to the interest brought about by the diagnostic problems of this disease. Lastly postural headache and its prevalence in the general population is examined. Various physiopathological aspects of this form (stress, psychosocial events) are evaluated.


Clinical trials with bisphosphonates.

Clinical trials with bisphosphonates. Bisphosphonates are nonbiodegradable pyrophosphate analogs that are capable of inhibiting bone resorption in vivo and in vitro. For this reason they have been used as effective therapeutic agents in several conditions characterized by increased bone turnover, including Paget's disease, hypercalcemia of malignancy, and metastatic bone disease. More recently, bisphosphonates have been proposed for the treatment and prevention of bone loss in several forms of osteoporosis. Etidronate, the first bisphosphonate to be used in clinical trials, has been found to increase vertebral bone mineral mass in osteoporotic patients. However, the gain in bone mass reaches a plateau after 1-2 years of treatment, with no further increase thereafter. No positive effect on osteoporotic fracture rate has been clearly demonstrated. Moreover, etidronate has been shown to impair bone formation and mineralization at therapeutic doses. Newer, more potent bisphosphonates selectively inhibit bone resorption without impairing bone histology and mechanical strength. Pamidronate has been shown to increase vertebral bone mass in patients with steroid-induced osteoporosis and involutional osteoporosis. In the latter group, this increase did not plateau and was found to be sustained for at least 4 years. However, pamidronate use is associated with relatively poor gastrointestinal tolerability. The use of another agent, clodronate, has been limited by the possible link with the onset of hematologic malignancies. Alendronate is another agent which in studies to date has been found to increase vertebral bone mass in postmenopausal patients. Alendronate also seems to be more potent and better tolerated than pamidronate.(ABSTRACT TRUNCATED AT 250 WORDS)


[Hemicrania: primary headache].

[Hemicrania: primary headache]. Migraine is the commonest form among the so-called primary headaches and the description of its clinical picture is lost in the mists of time. On the contrary, headaches of organic origin have only recently received a proper nosological individuation thanks to the modern technological progress achieved in the field of medicine. The migraine crisis, both with and without aura, is so typical in its clinical features that it does not require subtle instrumental methodologies to be diagnosed. In most cases a careful anamnesis provides all the elements necessary to formulate a precise diagnosis: the positive family history, the time and mode of onset of crises, the nature of head pain, the chronological stages are quite constant whatever the trigger factor may be. Some other considerations add further evidence to the primitivity of migraine. First of all, migraine can be relieved by quite structurally different drugs with different mechanisms of action. In addition, no significant relationship has ever been found between migraine and other pathologies.


[The pathogenetic bases of hemicrania].

[The pathogenetic bases of hemicrania]. Studies of regional cerebral blood flow in migraine with aura have shown that the aura phase is associated with hypoperfusion in the cortical area which relates topographically to the clinical symptoms. Thus, the previously hypoperfused area becomes hyperperfused. However, there is no strict association between hyperperfusion and headache. The mode of hypoperfusion propagation recalls the circulatory manifestations of experimental cortical spreading depression. In addition, there are no focal cerebral blood flow abnormalities in migraine without aura. During the headache phase of migraine, dilation of both the large extra- and intracranial arteries takes place. A bulk of biochemical evidence has suggested that the pain in migraine is caused by blood vessels which are dilated and sensitized by circulating pain-producing substances e.g. bradykinin, serotonin and histamine (sterile inflammation). Recently, perivascular trigeminal fibres (trigeminovascular system) which, when stimulated, release sensory peptides (substance P and the calcitonin gene-related peptide) capable of provoking marked vasodilation and plasma extravasation (neurogenic inflammation) have been identified. Thus, the activation of the trigeminovascular system is probably involved in the vasodilatative and nociceptive phenomena of the migraine attack. The finding of a reduced endorphinergic brain tonus in migraine patients supports the hypothesis of a central nociceptive derangement in migraine. Nonetheless, the exact relationship between vasodilation and headache remains to be defined. However, the potent antimigraine effectiveness of sumatriptan--an agonist of the serotonin receptors which selectively constricts dilated arteries during the migraine attack--once again stresses the fact that serotonin is probably the crucial factor in the link between vasodilation and headache.


[Plasma and tissue antibiotic concentrations: what is their prognostic value?]. Pharmacokinetic studies of antimicrobial agents usually involve the determination of tissue distribution, since most infections develop outside the vascular compartment. Comparative analyses of the results are often complicated by their extreme variability, depending on the various types of methods used, the type of antimicrobial agent, and the characteristics of the various tissue compartments. In general, concentrations in tissue and body fluids with no barriers to penetration of drugs are fairly predictable from blood levels in relation to simple diffusion through capillary pores. This is valid for hydrosoluble drugs such as the beta-lactams as well as liposoluble drugs like the quinolones and most of the macrolides. Thus we may derive, in a predictive sense for the clinical results, both the hematic and interstitial fluid pharmacokinetic profile of the above molecules, which are useful in relation to the MIC or MBC of extracellular pathogens. For molecules such as azithromycin, in which tropism is mainly intracellular, hematic levels do not reflect those which are therapeutic in the infection site. In tissues and body fluids with barriers to the penetration of drugs, such as the central nervous system, eye and bronchial secretions, it is always useful and especially for any new antimicrobial agent to determine the concentrations which are achieved in relation to both dosage and pathological state of the tissue compartment. The significance of the chemotherapeutic formula which gives substantial predictive value of the therapeutic efficacy at the level of blood concentrations in relation to minimal inhibitory concentration of sensitive bacteria, needs to be critically re-evaluated in light of the pharmacokinetic behavior of newer antimicrobial agents.


[Noci-autonomic-affective automatism, the physiopathological essence of hemicrania. The serotonin theory as a guiding principle in the labyrinth of interpretations]. After thousands of painful long-lasting migraine or extremely violent cluster headache attacks no one has yet traced histological inflammatory or degenerative alterations of the interested tissues able to explain such dreadful pain. Therefore it has seemed logical to include these pains among the unjustified aimless, non finalized types of pain. Furthermore, clinical characteristics of automatism, explosiveness and the course of these pains resemble other aimless pains like those of organic deafferentation (phantom pain) which appear in a desensitized limb after denervation or even in amputated subjects. Intense and long lasting pains in opioid abstinence, mainly located in the chest and in the hip, also have all the characteristics of aimless pain. Idiopathic cephalic pain, together with deafferentation or opioid-abstinence pain, seems to be due to a dysafferentation which, through different channels, follows an analogous mechanism. This mechanism seems to be due to a deficit of autoanalgesia which in both organic deafferentation (phantom limb) and in opioid-abstinence can be related to the disuse of afferences' modulation. In idiopathic headache such a failure of autoanalgesia is likely to be due to a genetic, idiopathic mechanism. Headaches are characterized by a clear deficiency of autoanalgesia which may manifest itself not only at the level of the cephalic segment, which is so rich in afferences, but it may even involve the whole body. Even if pain is the compulsory phenomenon to diagnose headache, one must consider that migraine is a symptomatic triad in which vegetative and emotional phenomena also emerge. These phenomena are interindependent and not interdependent as each of them may appear as a first manifestation of an attack; one must therefore consider the possibility of a "unicum movens". Serotonin was taken into consideration because of its action which interests all or nearly all vegetative-emotional pain transmitting pathways. Today's identification of four types and various sub-types of 5-HT receptors has revealed the extraordinary eclecticism of this transmitter which within migraine's clinical expression underscores that migraine sufferers are characterized by a marked sensitivity to all the drugs capable of acutely or chronically interacting with serotonin metabolism and binding with many serotonin receptor types and sub-types. So even if the migraine sphinx still proposes its enigma, researchers--with their incurable curiosity--may not only find more and more accurate and effective medication for many human beings but also start penetrating a mystery, a great challenge to human imagination.


[The etiology of bacterial respiratory infections in adulthood]. Upper and lower respiratory tract infections are the most frequently reported pathologies both in children and in adults. In particular, the most common clinical pictures are otitis media, principally chronic, and acute and chronic sinusitis. The microbiology of otitis media, which has been investigated thoroughly in the last year, confirmed a bacterial origin in 90% of the cases (while the incidence of the viral form is low). The etiologic agents responsible for chronic infections are Gram-negative bacteria, staphylococci and anaerobic bacteria, while in the acute forms, which are rare in adults but frequent in children, the responsible pathogens are principally Haemophilus influenzae, Streptococcus pyogenes and Branhamella catarrhalis. Among the etiologic agents of chronic sinusitis, which, from a clinical point of view, is the most frequently observed, there are staphylococci, Streptococcus pyogenes and many anaerobic bacteria. As regards lower respiratory tract infections, chronic bronchitis is the most frequent pathology in adults. But, in this case, the etiologic agents are more difficult to identify. Analysing the above-mentioned clinical pictures and their relevant etiologic agents, among the different therapeutic proposals, one of the most innovative and recent is represented by azithromycin. Azithromycin is the first of a new class of macrolides termed "Azalides" which shows particular microbiological and pharmacokinetic characteristics. As compared to other macrolides, it has an higher distribution volume, a longer half-life, a deeper intracellular penetration and a wider spectrum of action. It is rapidly concentrated in leukocytes PMN, monocytes, macrophages and fibroblasts and slowly released by these cells to the infected tissues. Azithromycin concentrations in tissues and sites of infection can be increased by the phagocyte uptake.(ABSTRACT TRUNCATED AT 250 WORDS)


[The clinical problems of bacterial infections of the upper respiratory tract]. ORL inflammations present particular pathogenetic and clinical characteristics due to the close anatomic and physiological connection among them and to their particular anatomic characteristics. The ear communicates with the rhinopharynx through the Eustachian tube, and this relationship explains how bacterial infections of the upper respiratory tract can cause otitis and how tubal dysfunctions are often responsible for chronic ORL infections, also favoured by the microcavity structure of the middle ear and the mastoid bone. Also the macro- and microcavity anatomic structure of the paranasal sinuses favours chronic infectious diseases, and their adjacency with the endocranial and facial structures justifies the secondary complications of these infections. The palatine tonsil and other structures of the Waldeyer's lymphatic ring play an important immune role in the first respiratory and digestive tract, and infections occurring in these structures (for example, streptococcal infection) can cause pathologies such as rheumatic disease. Other ORL infections are connected to immunodeficiency or atopy.


[Antibiotic therapy in bronchopulmonary infections]. Because of difficulties in accurately determining an etiologic diagnosis, the ideal treatment for lower respiratory tract infections remains questionable. Suggested regimens are made on the basis of clinical and epidemiological data. However, the single most common pathogen responsible for pneumonia remains Streptococcus pneumoniae. Atypical pneumonia in younger patients is best treated with macrolides. Older patients without debility or immunodepression are best treated with amoxycillin-ampicillin, second generation cephalosporins or cotrimoxazole, on the basis of local susceptibility patterns of microorganisms. In the treatment of acute bacterial bronchitis in chronic bronchial disease, most antimicrobial agents with activity in vitro against Haemophilus influenzae and Streptococcus pneumoniae are clinically efficacious. Among new pathogens, the importance of Chlamydia pneumoniae is variable according to the studies, and Moraxella catarrhalis was considered almost exclusively responsible for purulent exacerbations of chronic bronchitis. Therapy for empiric treatment of nosocomial pneumonia must ensure coverage for aerobic Gram negative bacilli: the most frequently used includes a semisynthetic penicillin plus an aminoglycoside, but monotherapy with newer broad-spectrum antibiotics (imipenem, ceftazidime, ciprofloxacin, timentin, etc.) seems to be equivalent to combination regimens. The lung is the most common target organ for infectious complications in immunocompromised patients but the diagnostic methods employed in the traditional work-up of pneumonia are often of little or no use in this setting. By far the two most useful clues to management of pneumonia in the immunocompromised host are the underlying host defect and the radiographic pattern of the lung infiltrate.(ABSTRACT TRUNCATED AT 250 WORDS)


[Hypertension and cardiovascular damage]. All major randomized trials of antihypertensive therapy have used cardiovascular events as endpoints. This approach has provided important information, but has also led to a few inappropriate conclusions. In particular, no sound information is available on the ability of antihypertensive therapy to prevent the cardiovascular lesions upon which events are superimposed. However, particularly in mild to moderate hypertensives, the primary goal of therapy is prevention of cardiovascular lesions rather than prevention of premature death. Sensitive and quantitatively reliable methods for evaluation of organ damage in hypertension are now available. For instance, the quantitative evaluation of coronary plaques has recently been employed in therapeutic trials of coronary artery disease; non-invasive methods (such as quantitative ultrasonography of carotid artery walls) can be employed in large trials of antihypertensive therapy to answer the question whether some class of antihypertensive drugs, such as calcium-antagonists) may be more effective in prevention and/or regression of atherosclerotic plaques than traditional antihypertensive agents.


Studies on the minimum requirements for in vitro "cure" of tumor cells by cytotoxic T lymphocytes. DBA/2-derived P815 tumor cells form progressively growing tumors when injected into the anterior chamber (AC) of normal BALB/c eyes. By contrast, P815 cells injected into the subconjunctival (SCon) space of BALB/c mice are promptly rejected. Despite these very different outcomes, both AC and SCon tumors elicit an equal clonal expansion of tumor-specific precursor cytotoxic T cells (pTc) within the draining cervical lymph nodes. Moreover, tumor-specific pTc migrate systemically and infiltrate both AC and SCon tumor-containing eyes. The present study explores the hypothesis that pTc generated in response to AC P815 tumors are less efficient "killers" than their SCon counterparts. To address this issue pTc were restimulated in vitro in the presence of exogenous lymphokines to initiate the terminal differentiation of pTc into fully functional cytotoxic T cells (Tc). Then an assay was developed to evaluate the capacity of AC and SCon CD8+ Tc to eliminate P815 tumor cells growing in vitro, i.e., achieve a "cure." This assay established the day tumor cells were completely eliminated (cure), as well as the rate of tumor cell removal. Using this approach it has been learned that pTc harvested from AC and SCon tumor-bearing mice have the same potential for eliminating P815 tumor cells in vitro. Moreover, the efficiency of achieving an in vitro tumor "cure" was found to be remarkably enhanced if exogenous lymphokines, as a surrogate source of T cell "help," were included in the cultures at the time effector lymphocytes were added to the proliferating tumor target cells. This latter finding emphasizes the need for sustained availability of helper factors in order to maximize the efficiency with which Tc can eliminate tumor cells. We interpret the results to mean that successful rejection of tumors in vivo is likely to turn upon (a) arrival of tumor-specific pTc at the tumor site, (b) delivery of "help" that promotes the terminal conversion of these cells into Tc, and (c) sustained presence of "help" which promotes the efficiency of immune elimination of tumor cells. Since equally large numbers of P815-specific pTc enter AC and SCon tumor sites in BALB/c mice, the failure of rejection in the former may be due to an inability of the host to provide immediate and sustained local T cell help. Implications of these studies for the development of immunotherapies for intraocular tumors are discussed.


Murine Lyme borreliosis: route of inoculation determines immune response and infectivity. Outer surface protein A OspA is the major outer surface protein of B. burgdorferi, the causative agent of Lyme disease, and has been advocated as a vaccine candidate. It is recognized late or not at all in the course of human Lyme disease, but has been identified as a major antigenic epitope for the anti-spirochetal immune response in a number of experimental models of B. burgdorferi infection. We injected B.burgdorferi into mice and tested the appearance of immunoreactivity to OspA by Western blotting. Three routes of infection were studied; other variables investigated were inoculum size and isolate of spirochete and strain of mouse. OspA immunoreactivity, as determined by Western blotting, was readily elicited by injection of sonicates under almost any condition. Intraperitoneal or intravenous injection of infectious spirochetes, especially at infective inoculum sizes, or injection of the noninfectious B31 isolate by any route, resulted in OspA immunoreactivity. However, mice from the three strains tested infected intradermally did not develop significant OspA immunoreactivity, but instead developed strong responses to B.burgdorferi proteins of different molecular weights. These data suggest that during infection within the skin after intradermal inoculation, the OspA protein may be altered in some way to make it less immunogenic than when it is presented to the immune system under other circumstances.(ABSTRACT TRUNCATED AT 250 WORDS)


Regional dichotomy of idiotypic expression on senescent B lymphocytes in systemic and mucosal immune systems: selective changes in NPb and T15 idiotypes with aging. The age-associated changes in the expression of NPb and T15 idiotypes on antibody-secreting B cells in the spleen and bronchial lymph nodes (BLN) of mice following peritoneal immunization with (4-hydroxy-3-nitrophenylacetyl) acetic acid (NP) and phosphorylcholine (PC) as antigens were investigated. Changes in the NPb or T15 idiotype expressions were detected by the inhibition of anti-NP or anti-PC elispot-forming cells (SFC) with monoclonal (Mab) anti-idiotypic antibodies, Mabs Ac38 and Ac146, or AB1-2, respectively. We found that Mabs Ac38 and Ac146 significantly inhibited anti-NP IgM and IgG SFC responses in the spleen from 4-month old animals in a dose-dependent manner as opposed to a lack of effect on 24-month old splenic cells. There were no significant effects on anti-NP SFC responses in the BLN from either young or old mice. Similar results were obtained using Mab AB1-2 inhibition of T15 idiotype on anti-PC SFC responses in the spleen and BLN. No age-related changes in the gamma- and kappa-light chain contents of the secreted anti-NP antibodies by B cells in the spleen and BLN were observed. The kinetics of the appearance of endogenously auto-anti-idiotype blocked, hapten augmentable (HA) anti-NP IgM SFC responses revealed significantly higher percentages of HA SFC in the spleen and BLN, 14 days after immunization for both young and old mice. The findings suggest that the age-related changes in the idiotypic expression on antigen-specific antibody repertoires in the spleen and mucosa-associated tissues may be caused by the activation of subsets of B cells with different VH gene expressions.