Skin inflammation: reactive oxygen species and the role of iron.

Superoxide and hydrogen peroxide are reactive oxygen species (ROS) primarily produced by phagocytic cells as a consequence of the process of phagocytosis. This defensive role, may, however, become one of attack when production of ROS is excessive and overwhelms cellular scavenging systems. This happens in situations such as acute inflammation and results in host cell membrane damage, which is particularly prevalent in the presence of transition metal catalysts such as iron and copper. The skin is uniquely vulnerable to this attack being rich in polyunsaturated fatty acids and exposed to high oxygen tensions and ultraviolet light, both of which promote production of ROS. Additionally, the respiratory burst of infiltrating polymorphonuclear leukocytes and macrophages in inflamed skin will produce high local levels of superoxide that can release "catalytic iron" from storage proteins such as ferritin. The role of iron and ROS in the pathogenesis of inflammatory skin disease is discussed as is the possibility of novel therapeutic strategies based on their removal.


Noncollagenous (NC1) domain of collagen VII resembles multidomain adhesion proteins involved in tissue-specific organization of extracellular matrix.

Type VII collagen (C7) is a stratified squamous epithelial basement membrane protein composed of three identical alpha chains, each consisting of a 145-kDa amino-terminal noncollagenous (NC1) domain and a 145-kDa carboxyl-terminal collagenous domain. Morphologic and biochemical studies have shown that tissue-specific aggregates of C7 dimers called anchoring fibrils may contribute to epithelial basement membrane organization and adherence by interacting with extracellular matrix (ECM) proteins such as type IV collagen. In this study, we cloned a cDNA encoding most of the NC1 domain of C7. The deduced amino acid sequence revealed motifs characteristic of multidomain ECM proteins that contribute to the tissue-specific organization of ECM including a region of 7 1/2 sequential fibronectin type III (Fn III) homology repeats, a potential collagen-binding region homologous to the A domain of von Willebrand factor (vWf) and an RGD sequence. A purified C7 fusion protein containing these motifs specifically bound to type IV collagen in a functional assay. These results suggest that regions within the NC1 domain of C7 mediate interactions with lamina densa and dermal ECM proteins including type IV collagen. Structural mutations and autoepitopes in these regions may represent mechanisms for the development of defective basement membrane organization and adherence in genetic and autoimmune forms of epidermolysis bullosa.


In vitro model of essential fatty acid deficiency.

The polyunsaturated fatty acids linoleic acid (18:2, n-6) and arachidonic acid (20:4, n-6) are essential for normal skin function and structure, both as eicosanoid precursors and as components of lipids forming cell membranes. Adult human keratinocytes grow optimally in serum-free medium (MCDB 153) that contains no fatty acids. These keratinocytes expand rapidly and produce normal epidermis upon in vivo grafting. Analysis of lipid extracts of epidermis and of cultured keratinocytes was done to determine the fatty acid composition of cells grown in essential fatty acid (EFA)-deficient medium. Gas chromatography and high-performance liquid chromatography analyses were done of the fatty acids in the entire cell and in a thin-layer chromatography separated fraction containing those lipids that form cellular membranes. Comparison of snap-frozen epidermis and epidermal basal cell suspensions to passage 1 to 4 cultures shows that the cells are in an extreme essential fatty acid-deficient state by the first passage. The amount of the saturated fatty acids 16:0, 18:0, and 14:0 is unchanged by culture. The polyunsaturated fatty acids are found to be significantly decreased, the cells balancing their lack with a significant increase in the relative abundance of the monounsaturated fatty acids, 18:1 and 16:1. Greater than 85-90% of the fatty acids was found in lipids associated with membranes and no unusual fatty acids were detected. Because the serum-free medium is fatty acid free and the cells cannot synthesize essential fatty acids, the rapid division of the cells results in the predominance of an extreme EFA-deficient cell type. The essential fatty acid-deficient keratinocyte is an excellent adult, normal epidermal cell model that can be used to study EFA deficiency and the effect of the eicosanoid and fatty acids on cell function and structure.


Collagenase in wound healing: effect of wound age and type.

Collagenase is believed to be important for cell migration and collagen remodeling during tissue repair and regeneration. We have investigated collagenase concentrations in different types of surgically inflicted wounds in pigs. Collagenase was extracted from tissue homogenates of wounds by heating to 60 degrees C for 6 min in 0.1 M CaCl2. The molecular weight of latent collagenase was about 52 kDa. Activated collagenase produced the characteristic 3/4 fragment of collagen. Collagenase was assayed by the use of radiolabeled telopeptide-free collagen. To detect maximal collagenase activity, extracts were reduced and alkylated to destroy inhibitors, then activated with aminophenylmercuric acetate. Sutured incisions showed peak collagenase content on postoperative day 1 and thereafter steadily declining concentrations. Granulation tissue from non-sutured large defect full-thickness wounds showed high collagenase content on postoperative day 5 and then a sharp decline to day 7 followed by a slowly declining curve to postoperative day 21. Partial-thickness wounds exhibited a different time course, with collagenase increasing to peak concentrations on postoperative days 3-5; however, a large proportion of the detected collagenase was due to the adherent scab. By day 7 collagenase concentrations approached the low concentrations of normal skin when epithelialization was complete and the scab rejected. In general, collagenase shows an early maximum and then declines with postoperative time, with the sharpest decline occurring when epithelialization is complete.


Effect of aging and habitual sun exposure on the genetic response of cultured human keratinocytes to solar-simulated irradiation.

Aging and chronic sun exposure are known to be associated with decreased cutaneous immune function, changes in the balance between epidermal proliferation and differentiation, and a greatly enhanced risk of photocarcinogenesis. However, their specific effects on the response of human keratinocytes to ultraviolet (UV) irradiation are unknown. We therefore asked whether aging and photoaging modulate the response at the mRNA level to UV-inducible genes implicated in immunomodulation and/or growth control. Cultured human keratinocytes derived from newborn, young adult, and old adult donors were exposed to a single physiologic dose of solar-simulated UV or sham irradiation and harvested at 1, 4, 8, 24, and 48 h post-irradiation for northern blot analysis. Specific mRNA was detected using cDNA probes encoding the proto-oncogenes c-fos and c-myc and the growth-arrest and DNA damage (GADD153) gene, all recently shown by our laboratory to be modulated by UV in newborn keratinocytes; interleukin (IL)-1a, IL-1b, and the IL-1 receptor antagonist (IL-1ra), two keratinocyte cytokines and their competitive inhibitor, implicated in the immunomodulatory effect of UV; and SPR2, a recently cloned gene known to be induced during normal keratinocyte differentiation and by lethal UV-C irradiation. Our data suggest that aging alone strikingly increases the baseline expression of SPR2 and IL-1ra but has relatively little effect on the response to UV for the other genes examined. In contrast, the combination of aging and habitual sun exposure, so-called photoaging, markedly increases c-fos inducibility and decreases baseline expression of SPR2 and IL-1ra relative to that in cells from sun-protected skin of the same donors. The implied alterations in signal transduction and differentiation state observed in cells derived from habitually sun-exposed sites of old adults may explain in part the predisposition to photocarcinogenesis in photoaged skin.


Demonstration of clonal immunoglobulin gene rearrangements in cutaneous B-cell lymphomas and pseudo-B-cell lymphomas: differential diagnostic and pathogenetic aspects.

Twenty-five patients with a benign or malignant cutaneous B-cell lymphoproliferative disease, including seven cutaneous pseudo-B-cell lymphomas, eight primary cutaneous B-cell lymphomas (CBCL), and 10 secondary cutaneous B-cell lymphomas, were investigated for the presence of clonal immunoglobulin (Ig) gene rearrangements using Southern blot hybridization analysis. The selection of pseudo-B-cell lymphomas was based on the presence of polyclonal light-chain expression with immunohistochemical analysis. All cases of CBCL demonstrated monotypic light-chain expression or absence of detectable Ig on CD20+ B cells. Clonal rearrangements of one or more Ig genes were demonstrated in four of seven cases of cutaneous pseudo-B-cell lymphomas, six of eight cases of primary CBCL, and in all cases of secondary CBCL. The observation that cutaneous pseudo-B-cell lymphomas as defined by immunohistochemical criteria often contain occult monoclonal B-cell populations implies that differentiating between pseudo-B-cell lymphomas and CBCL is not always possible by means of gene-rearrangement analysis. These findings may support the concept that cutaneous pseudo-B-cell lymphomas and primary CBCL are part of a continuous and progressive spectrum of B-cell lymphoproliferative skin disorders.


Membrane structural abnormalities in the stratum corneum of the autosomal recessive ichthyoses.

Congenital ichthyosiform erythroderma (CIE) and classic lamellar ichthyosis (LI) are autosomal recessive disorders of cornification (DOC), distinguished previously by clinical, histologic, ultrastructural, and cell kinetic criteria. Whether there is further heterogeneity within the CIE group is uncertain. To address the issue of genetic heterogeneity, and to study the pathogenesis of these DOC, skin biopsies from eight CIE, three LI, and six normal subjects were assessed by electron microscopy, including ruthenium tetroxide postfixation with optical diffraction, to visualize and quantitate intercellular membrane domains. We found abnormal lamellar bodies in CIE and distinctive alterations in intercellular lamellar bilayer architecture among patients with CIE and three patients with LI. Two biopsies from two patients at different sites demonstrated the consistency of these findings. Moreover, in both CIE and the three LI patients, desmosomes persisted throughout the outer layers of the SC, indicative of impaired degradation. Our ultrastructural observations support the previously reported phenotypic distinction between CIE and LI, and the further likelihood of genetic heterogeneity within CIE. However, these studies do not support the division of the autosomal recessive ichthyoses into three subgroups based upon cytosolic structural abnormalities. Finally, these studies provide new insights into the pathogenesis of the autosomal recessive DOC.


Comparison of calmodulin gene expression in human neonatal melanocytes and metastatic melanoma cell lines.

The qualitative and quantitative expression of three calmodulin genes (CAM I, CAM II and CAM III) was characterized in human neonatal melanocytes and metastatic melanoma cell lines in the absence and presence of serum, other growth modulators, and/or 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Results indicated that the qualitative expression in melanocytes was the same as that of melanomas, that is, CAM I gene expressed two transcripts, 4.4 kb and 2.1 kb, whereas CAM II and CAM III expressed one transcript each, 1.95 kb and 2.37 kb, respectively. Differential quantitative expression was seen particularly with CAM I. The average levels of both CAM I transcripts in melanomas were less than one-half those of melanocytes. Serum and other growth modulators (including Ca++, isobutyl methyl xanthine, bovine pituitary extract, and insulin) enhanced CAM I and CAM II gene expression in melanocytes; in contrast, the net effect of serum in melanomas was to decrease expression of CAM I and CAM III. This effect was most prominent in melanoma C81-46C. TPA markedly inhibited expression of all three CaM genes in melanocytes; however, in melanomas the net effect of TPA was to increase their expression. CAM I in melanoma C81-46C was the most sensitive to TPA stimulation.


Three-dimensional reconstruction and stereometric analysis of Langerhans cells in mouse epidermis.

In the presented studies stereometric analysis and spatial reconstruction was performed on two Langerhans cell (LC) types. One was free of LC-I and the other contained LC-II Birbeck granules in the perinuclear space. The presented stereometric analysis demonstrated significant differences between the so-distinguished two cell types. Differences were observed not only in the number and distribution of Birbeck's granules but also in the areas of smooth and rough endoplasmic reticulum, in the area of vesicles surrounding Golgi apparatus, in the volume of cisterns of the apparatus, and in the ratio of cell nucleus area to its volume. Differences noted between the two cell types were of quantitative character. They might result from different stages of differentiation of the cells from their precursors in the epidermis or from distinct functional stages of the cells.


Mitotic activity in the dermis of mice during graft versus host disease: the role of fibroblast replication in dermal fibrosis.

When immunocompetent cells are transferred to an allogeneic, immunologically compromised host a complex series of cellular events are initiated, referred to as graft versus host disease. This results in widespread organ damage with fibrosis being a prominent feature. The pathologic fibrosis may result from an increase in fibroblast numbers, an increase in collagen produced from individual fibroblasts, or a combination of the two processes. The relative contribution of fibroblast replication to the pathologic fibrosis seen in graft versus host disease has not been directly determined previously, and this is the main object of this paper. Graft versus host disease was induced by the transfer of lymphoid cells from B10D2 mice to irradiated Balb/c recipients. In order to study the mitotic activity of dermal cells following bone marrow transplantation, a thymidine anologue, bromo-deoxyuridine (BrDU), was administered to mice using an osmotically driven, implantable infusion device. The labeled cells were visualized immuno-histochemically and studied at weekly intervals. There is intense mitotic activity in the basal layer of the epidermis and the acrosyringal epithelium from the second week. Evidence of increased mitotic activity in the epidermis persisted until the fifth week post-transplantation. Fibroblast replication was seen from the end of the third post-transplant week. Dermal collagen deposition also occurred at this time. Peak mitotic activity was present at the end of the fourth week and was less pronounced by the fifth week. It was especially evident in the upper dermis where the developing collagen layer was being deposited. To our knowledge this is the first direct demonstration of fibroblast proliferation in an immunologically mediated fibrotic disorder. It is concluded that fibroblast replication is an important mechanism leading to the pathologic fibrosis seen in graft versus host disease and, by analogy, probably other types of immunologically mediated fibrosis.


Topically applied verapamil hydrochloride inhibits tuberculin-induced delayed-type hypersensitivity reactions in human skin.

Calcium channel antagonists have been reported to possess inhibitory effects on lymphocyte migration and activation in vitro, and on cell-mediated immune reactions in the skin of experimental animals. We have therefore studied the effects of topically applied 8% (w/v) verapamil hydrochloride in propylene glycol on tuberculin-induced delayed-type hypersensitivity reactions in the skin of normal human volunteers. There was significant inhibition of the tuberculin reactions by the verapamil preparation compared to vehicle controls, as determined by forearm skin fold thickness measurement and assessment of the density of mononuclear cell infiltrates in skin biopsies. The precise mechanism of action of verapamil hydrochloride remains unclear, but could include effects on T-cell migration and activation, on antigen-presenting cells, and/or on other cells. The potential for the use of topical calcium channel antagonist preparations in inflammatory skin diseases warrants further study.


Molecular mechanisms of human melanocyte attachment to fibronectin.

In this report we show that fetal and neonatal melanocyte attachment to fibronectin (FN) is inhibited by antibodies to the beta 1 integrin subunit, suggesting a role for these molecules in melanocyte attachment to FN. The VLA-5 integrin was shown to be the predominant receptor for fetal melanocyte attachment to FN, in contrast with neonatal melanocytes in which the very late antigen (VLA)-5, VLA-3, and alpha v integrins each contributed to melanocyte attachment to FN. Peptides containing the arginyl-glycyl-aspartyl-serine (RGDS) sequence inhibited fetal and neonatal melanocyte attachment to FN by a maximum of 48% and 85%, respectively. The almost complete inhibition of neonatal melanocyte attachment to FN by RGDS-containing peptides suggests that the central cell-binding domain of FN is the primary recognition site for neonatal cell attachment to FN. Fetal and neonatal melanocytes showed a concentration-dependent attachment to two proteolytically derived fragments of the FN molecule: a 75-kD fragment, which contains the central cell-binding domain, and 33/66-kD fragments of the FN molecule, which encompass the heparin-binding domains V and VI. Antibodies to the beta 1 subunit inhibited fetal and neonatal melanocyte attachment to the 33/66-kD fragments by a maximum of only 15% and 24%, respectively, suggesting that other, non-integrin, receptors are involved in melanocyte recognition of this portion of the FN molecule. We propose that human fetal and neonatal melanocytes attach to FN by different complements of receptors and ligand target sequences, and that these differences may direct melanocyte interactions with FN during development.


Effective enrichment of murine epidermal Langerhans cells by a modified(mismatched) panning technique.

A method for the enrichment of murine epidermal Langerhans cells (LC) is described in detail. It is based on positive selection of LC from pre-enriched fresh or cultured epidermal cell suspensions derived from ear skin by a modified panning technique. The method uses the interspecies cross-reactivities of anti-immunoglobulin antibodies: when LC in an epidermal cell suspension are labeled with mouse anti-major histocompatibility complex (MHC) class II antibodies they bind to petri dishes coated with anti-rat immunoglobulin antibodies. We therefore call this method "mismatched panning." After rinsing off non-adherent cells, the adherent LC can easily be dislodged by adding excess amounts of rat immunoglobulins, which effectively compete with the LC-bound mouse anti-MHC class II antibodies for binding to the petri dish. Using this modified panning technique, both fresh and cultured LC could be enriched up to more than 90% purity. From one ear, 2.0-3.0 x 10(4) fresh LC and 3.0-4.5 x 10(4) cultured LC could be obtained. Of all LC present in a primary, unenriched epidermal cell suspension, 40-60% were recovered when panned immediately after isolation of the epidermal cells and 50-75% when panned after 3 d of epidermal cell culture. Viability of panned LC was consistently more than 90%. Antigen presenting and T-cell-stimulating capacity of LC and responses to the cytokines granulocyte/macrophage colony-stimulating factor and tumor necrosis factor-alpha were not impaired by this panning procedure. The major advantage of this method compared to pre-existing panning techniques is the ease with which adherent LC can be dislodged from the panning dishes. Because the elution procedure is very gentle, virtually all panned LC are viable. As a consequence, good yields of highly enriched LC can be obtained in a reasonable time.


Preclinical system for evaluating topical podofilox treatment of papillomas: dose-response and duration of growth prior to treatment.

The objective of the present study was to assess the utility of Shope rabbit papillomas as an animal model system for studying topical podofilox treatment and to evaluate dose-response relations and influence of duration of papilloma growth prior to treatment. New Zealand White rabbits received inoculations of cottontail rabbit papillomavirus (CRPV) virions of two dilutions at four sites total on the dorsum. Two papillomas on the left side were treated with podofilox (Oclassen Pharmaceuticals, Inc., San Rafael, CA). The drug was given topically twice each day, 5 d per week, for 21 d. We evaluated the effects of drug dose and the duration of papilloma growth prior to treatment. Results indicated that treatment beginning on day 28 with both 0.5 and 2.5% (w/v) podofilox inhibited papilloma growth, but 5.0% was more effective. In a separate experiment, papillomas were treated at 7, 21, or 60 d after virus inoculation. At 7 d, the untreated lesions were latent (not visible). At 21 d after infection, they were about 2.5 mm in diameter. At 60 d, papillomas were about 25 mm. Treatment with 5.0% podofilox beginning on any of those days strongly inhibited papilloma growth. Neither Southern blots nor PCR detected CRPV DNA in cured sites of previous virus infection. Antibody production to CRPV virion was not affected by drug treatment. 5.0% podofilox irritated normal skin adjacent to papillomas as evidenced by inflammation, induration, and superficial erosion. However, healing was satisfactory and no scarring resulted. We concluded that the Shope papilloma was a good model system for studying podofilox treatment because the lesions responded to drug across a broad range of drug concentrations and papilloma sizes.


Use of the polymerase chain reaction in quantification of interleukin 8 mRNA in minute epidermal samples.

Quantitative studies of cytokine gene expression in vivo are necessary in order to properly describe the cytokine network and to elucidate its role in skin inflammation. Ideally, one should be able to follow cytokine gene expression in epidermal, dermal, and blood compartments. However, such studies are limited by small amounts of available material. Here we report a polymerase chain reaction (PCR) cDNA amplification protocol useful for quantification of specific mRNAs in small skin samples. We found that analysis of dilution series of each sample permitted establishment of quantitative PCR amplification conditions using only picogram to nanogram amounts of total RNA. Cytokine mRNA amounts could then be measured relative to an internal standard species, co-reverse transcribed, and co-amplified with the cytokine species as a measure of cDNA input. Large numbers of samples can be screened rapidly with initial short dilution series identifying cytokine-positive samples and the correct dilution range for each, followed by closer analysis in this range. Epidermal samples obtained through curettage of a small skin area, 2-mm dermal biopsies from the scraped sites, and a few blood drops from the biopsy sites all yielded sufficient RNA for analysis by this protocol. Any mRNA of known sequence can be studied. We analyzed interleukin 8 mRNA levels in more than a hundred epidermal samples from patients and normal test persons and found a variation over several orders of magnitude that seemed to follow the degree of inflammation of the skin.


Constitutive synthesis of a 92-kDa keratinocyte-derived type IV collagenase is enhanced by type I collagen and decreased by type IV collagen matrices.

Human keratinocytes synthesize interstitial collagenase, a 72-kDa gelatinase, and a recently described 92-kDa gelatinase/type IV collagenase. We examined the synthesis of this novel enzyme by basal keratinocytes apposed to plastic, basement membrane collagen (type IV), and interstitial dermal collagen (type I). Samples of conditioned medium were electrophoresed on a 10% polyacrylamide, gelatin-ladened zymogram. Protein bands with gelatin-cleaving properties were identified by clarification of the gel and quantified by densitometry. A 92-kDa band had marked gelatinolytic activity and increased in culture over 72 h. The identification of this 92-kDa band as type IV collagenase was demonstrated by Western immunoblotting using monospecific antibody to the 92-kDa type IV collagenase. Keratinocytes apposed to type I collagen exhibited a threefold increase in the synthesis of the 92-kDa enzyme compared to cultures apposed to type IV collagen and a 1.5-times increase compared to plastic. The specificity of this enhancement was shown by constant levels of other proteins (e.g., the 72-kDa gelatinase). This study demonstrates that cell-matrix interactions modulate the synthesis of a recently described, keratinocyte-derived, 92-kDa gelatinase and that specific collagen types (I versus IV) have opposite effects upon the synthesis of this enzyme.


Selective expansion of V delta 1 + T cells from leprosy skin lesions.

T cells bearing gamma delta T-cell receptors (TCRs) are prominent residents of murine epidermis and appear to be important participants in the immune response to infection in human skin. The Mitsuda reaction in leprosy, induced by intradermal challenge with Mycobacterium leprae, provides an opportunity to study the cellular events that mediate a form of delayed-type hypersensitivity (DTH) in skin. T cells bearing gamma delta TCRs comprise a significant proportion of the T-cell population in these DTH reactions. Presently we have generated T-cell lines from Mitsuda reactions in vitro and compared their TCR repertoire to that found in situ. gamma delta T cells comprised 20-40% of lines derived from these skin lesions, but < 10% of lines derived from the peripheral blood of the same individuals. Flow-cytometric analysis of variable (V) chain usage in T-cell lines derived from skin lesions indicated that V delta 1 was predominant. Evaluation of the TCR repertoire using PCR indicated that V delta 1-J delta 1 and V gamma 2-J gamma P gene rearrangements were prevalent. In comparison, V delta 2-J delta 1 gene rearrangements predominated in situ. Furthermore, nucleotide sequence analysis of the V-J junction of one T-cell line revealed limited genetic diversity of the gamma delta TCR. These findings suggest that the V delta 1 subpopulation of gamma delta T cells in Mitsuda skin reactions selectively outgrows from leprosy skin lesions in vitro. Such V delta 1 + T-cell lines should be useful for determining the relevant antigens and restriction elements in this response to a pathogen in skin.


Molecular heterogeneity of the fourth component of complement (C4) and its genes in vitiligo.

In view of evidence suggesting vitiligo is an autoimmune disease, we investigated whether vitiligo is associated with inherited deficiencies of the fourth (C4) and second (C2) component of complement and with certain human leukocyte antigens (HLA). Analysis of functional activities of C4 and C2 in sera of patients with vitiligo (n = 42) showed that 17% of them had a heterozygous C4 deficiency and 5% had a heterozygous C2 deficiency. In the normal control group (n = 30), 3% had a heterozygous C4 deficiency and none had a C2 deficiency. C4 typing by Western blot analysis showed the frequency of the C4A*Q0 allele in the vitiligo patient group to be close to normal. However, the frequency of one C4B*Q0 allele was three times higher, and that of two C4B*Q0 alleles five times higher in the vitiligo patient group than the reported frequencies in normal control groups. Southern blot analysis of Taq1 digests of DNA using C4 and 21-hydroxylase probes showed that two patients with two C4B*Q0 alleles had a deletion of a 21-OHA-C4B segment. In the other patients, having one or two C4B*Q0 alleles, these null alleles probably occurred due to a loss of C4 gene expression. HLA analysis did not show any allelic association of C4A*Q0 or C4B*Q0 with any HLA antigen in vitiligo, but confirmed the previous findings of a negative association with HLA-DR3 and a positive association with HLA-DR4. These results suggest that abnormalities of the C4B gene and the above-mentioned associations with HLA antigens may be some of the risk factors in vitiligo.


Characterization of the oxidative stress initiated in cultured human keratinocytes by treatment with peroxides.

Cultured human keratinocytes were treated with H2O2, Fe++/Fe , H2O2 + Fe++/Fe , t-butylhydroperoxide (tBHP), or cumene hydroperoxide (CHP). Fe++ +/- Fe was without effect on cell viability. Neither CHP, tBHP, nor H2O2 at 200 microM led to alteration of trypan blue exclusion, but with 700 microM CHP or tBHP there was uptake of trypan blue after 20 min and lysis of cells beginning at 4 h of treatment. Lysis occurred even if the organic hydroperoxide was removed from the media after 1 h. Treatment with 700 microM H2O2 resulted in half of the cells becoming permeable to trypan blue by 60 min, but > 80% of the cells remained intact and functional, and eventually recovered their impermeability to trypan blue. No concentration of H2O2, tBHP, or CHP produced significant thiobarbituric acid (TBA)-reactive material, and Fe++/Fe , H2O2 + Fe++/Fe , and CHP + Fe++/Fe led to the formation of only small amounts of TBA-reactive material. This was attributed to a lack of polyunsaturated lipid in cells cultured in synthetic media. The activity of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is sensitive to oxidative damage and thus was used as an indicator of oxidative stress along with the ratio of reduced/oxidized glutathione (GSH/GSSG). Using these two criteria, we found that CHP or tBHP treatment led to an oxidative stress that was more protracted as compared with the effect of H2O2. The organic peroxides also led to depletion of total glutathione, an effect not found with H2O2. It was also found that H2O2 was more rapidly metabolized than the organic peroxides. In summary, cultured human keratinocytes treated with peroxides underwent a number of changes, which included inactivation of GAPDH, a decrease in the ratio GSH/GSSG, and a loss of trypan blue exclusion. However, as long as the duration of this oxidative stress was short, these changes were reversible and the cells survived. Prolonged oxidative stress led to irreversible damage and cell death. H2O2 was rapidly metabolized and relatively well tolerated by keratinocytes. On the other hand, organic hydroperoxides were metabolized more slowly and were lethal at sub-millimolar concentrations. The relative toxicity of organic hydroperoxides is hypothesized to be related to their non-polar nature.


Breast-cancer-associated stromelysin-3 gene is expressed in basal cell carcinoma and during cutaneous wound healing.

Ten cases of basal cell carcinoma (BCC), including nine of the nodulo-ulcerative type and one of the morphea-form type, were investigated for stromelysin-3 (ST3) gene expression by in situ hybridization. The ST3 gene, which codes for a putative matrix metalloproteinase expressed in stromal cells of invasive breast carcinomas, was also expressed in stromal cells of BCCs when they displayed active local invasiveness. ST3 RNA was specifically detected in fibroblastic cells of tumor areas exhibiting loss of peripheral palisading in cancer cell islands. This pattern of expression was characteristic of the ST3 gene and was not observed with any of the other matrix metalloproteinase genes tested. We suggest that ST3 gene expression, which was also observed in fibroblasts during cutaneous scar formation, corresponds to a normal wound-healing response that has been subverted in carcinomas.


The effects of recombinant human erythropoietin on the cell mediated immune response of renal failure patients.

Recombinant human erythropoietin (rHuEpo) is now widely employed in correction of the anemia of end stage renal disease (ESRD). Recent reports suggest that rHUEpo, in addition to its effect on CFU-E and burst-forming-unit-erythroid (BFU-E), may stimulate granulocyte/macro-phage production and pluripotential progenitors of the myeloid and monocyte lineage. Furthermore, there is now data which demonstrate that ESRD patients who received rHuEpo have enhanced cytokine production. Taken together, these observations suggest that the administration of rHuEpo may augment the diminished immune response of renal failure patients. To evaluate the effects of rHuEpo therapy on cell-mediated immunity in hemodialysis patients, a prospective controlled study was conducted. Two parameters of immune function were tested. One was natural killer cell (NK) activity, and the other proliferation in response to the T cell mitogen concanavalin A (Con-A). NK activity of the ESRD patients was comparable with that of normal controls at the start of the study and was unaffected by rHuEpo therapy. In contrast to this, anemic ESRD patients initially demonstrated impaired mitogen driven proliferation (initial stimulation index (S.I.) = 42.5 +/- 11.9) which significantly improved following rHuEpo therapy (final S.I. = 73.3 +/- 14.7, p < 0.05). The later value exceeded the mitogen response in less anemic ESRD patients who did not receive rHuEpo (initial S.I. = 60.7 +/- 16.5, final S.I. = 61.0 +/- 16.7), but did not reach values seen in normal controls. The data suggest that rHuEpo therapy may be associated with enhanced immune responses in patients with ESRD.


Cardiac neurosis: interactions between cardiac arrhythmias and symptoms using ambulatory ECG monitoring.

To investigate interactions between cardiac arrhythmias and subjective complaints and the background, ambulatory electrocardiographic (ECG) monitoring and a mental test [Cornell Medical Index (CMI)] were performed on 32 patients who complained of anxiety due to palpitation and/or tachycardic feelings without organic heart disease. The patients were classified into two groups according to the Holter ECG. In one group symptoms corresponded with cardiac arrhythmias (Group C; n = 15); and the other group lacked corresponding arrhythmias in spite of their significant symptoms (Group B; n = 17). From psychological view points, 65% of Group B and 40% of Group C patients showed grades III or IV of CMI tests, whereas only 5% showed grade III in normal volunteers (Group A; n = 20). Patients suffering symptoms without associated cardiac arrhythmias may have psychophysiologic backgrounds, at least in a part. It might be necessary in the treatment of these patients to pay attention to the factor of cardiac neurosis.


Effects of flunoxaprofen, a non-steroidal anti-inflammatory agent, on the cardiovascular system.

The effects of flunoxaprofen (FL) were investigated using anesthetized normotensive and spontaneously hypertensive (SHR) young and old rats. The animals were divided into two groups. One group received a non-steroidal anti-inflammatory agent at doses significantly inhibiting prostaglandin (PG)-cyclooxygenase; the other received no non-steroidal anti-inflammatory drugs. Oral pretreatment of anesthetized rats with flunoxaprofen (6.6, 11.5, and 19.9 mg/kg/day in the feed for 560 days) significantly increased the following responses: occlusion of the common carotid arteries, and reactivity to L-noradrenaline (NA) (i.v.), and angiotensin II (Ang) (i.v.). Pressor responses also increased in normotensive and in SHR rats treated with FL (20 mg/kg/day for seven days). The effects were more intense in the SHR rats. Moreover, oral pretreatment of normotensive rats with FL (20 mg/kg/day for seven days) partially yet significantly reduced the antihypertensive activity of etozolin (ET), a diuretic and antihypertensive drug. Our data confirm the important role of prostaglandins in the regulation of the cardiovascular system, and an increase in arachidonic acid metabolite biosynthesis with vasodilatory effects being part of the mechanism of action of some antihypertensive drugs.


Dramatic improvement with clonidine in acute pancreatitis with elevated catecholamine and cortisol plasma levels: report of five cases.

We report five cases of patients with acute pancreatitis (AP) resistant to conventional treatment who improved dramatically with clonidine. All patients showed greatly elevated plasma levels of noradrenaline (norepinephrine) (NA), adrenalin (epinephrine) (Ad) and cortisol (CRT), physiologic indicators of stress, which decreased abruptly upon initiation of clonidine therapy. The clonidine test performed in the patients showed a hyper-response in all, a reaction consistent with uncoping stress situations. Therefore, we postulate that stress might play a role in the pathogenesis of pancreatic inflammatory disease in these patients.


Tuberculous peritonitis with extraordinarily high serum CA125.

A 74-year-old woman who was admitted for the evaluation of ascites showed a marked elevation of serum CA125 level. Laparoscopy revealed the presence of numerous white modules (approximately 5 mm diameter) on the peritoneum suggestive of tuberculomas. A biopsy specimen showed epithelioid cells and Langhans-type giant cells. Following the administration of diuretics, the level of serum CA125 decreased concomitantly with the decrease in the volume of peritoneal fluid. The level of CA125 decreased further with the administration of antituberculosis agents. Clinical specimens were negative for mycobacteria. We conclude that the elevated serum level of CA125 was due to peritoneal tuberculosis and consequent ascites. Thus, tuberculous peritonitis should be considered in the presence of an unexplained, abnormally elevated serum level of CA125.


A long-term survivor with malignant thymoma accompanied with pure red cell aplasia.

A 56-year-old woman with malignant thymoma lived for 19 years after diagnosis and died of superior vena cava syndrome. An autopsy showed invasion of the thymoma to the right lung and metastasis to the left lung. The pathologic diagnosis was epithelial thymoma (spindle cell dominant) accompanied with pure red cell aplasia (PRCA). However, this was well controlled with steroid therapy. She was the longest survivor with malignant thymoma at this time. Her long-term survival may be the result of good control of the PRCA.


Paraspinal autonomic ganglion distortion due to vertebral body osteophytosis: a cause of vertebrogenic autonomic syndromes?

Preliminary histological investigation to determine whether vertebral body osteophytosis can compromise adjacent paraspinal autonomic structures. Histological sections of the L4 to S1 vertebral bodies were cut in the coronal plane at a thickness of 200 microns for morphological studies using transmitted light microscopy. Morphological evidence is presented which supports the hypothesis that vertebral body osteophytes appear to compromise adjacent paraspinal autonomic structures. Motion segment osteophytosis may affect viscera via the autonomic nervous system.


Intersegmental sagittal motion in the lower cervical spine and discogenic spondylosis: a preliminary study.

The object of this investigation was to identify any correlation between discogenic spondylosis and the type of motion (normal, hypomobility, hypermobility, paradoxical motion) found in the sagittal plane of the intervertebral motion units of the lower cervical spine. A case control study was performed from the files of 100 patients (ages 15-73) with cervical spine-related symptomatology at the Anglo-European College of Chiropractic Clinic. The cases were randomly selected from a cohort of patients with normal radiographic anatomy who attended the clinic from 1987-1990 and were known to have cervical spine neutral, flexion and extension lateral radiographs taken. Extended chi 2 was used to test the observed data. The findings from both the flexion and extension films suggested that intervertebral motion units with and without varying severities of discogenic spondylosis did differ with respect to the type of motion exhibited there (flexion: chi 2 = 39.399, p < .001; extension: chi 2 = 45.7424, p < .001). Intervertebral motion units which had discogenic spondylosis had a greater likelihood of exhibiting motion abnormalities (flexion: chi 2 = 5.665, p < .01; extension: chi 2 = 6.178, p < .01), and all types of motion seemed to be dependent on its severity (flexion: chi 2 = 16.464, p < .01; extension: chi 2 = 15.954, p < .02). In general, normal motion occurred approximately 60% of the time when there was absent or mild discogenic spondylosis and decreased precipitously as moderate and severe amounts of discogenic spondylosis appeared. In global cervical flexion, when there was either little or no discogenic spondylosis and abnormal motion was present, intersegmental hypermobility was predominant. Hypomobility became predominant overall as moderate and severe discogenic spondylosis was found. In global cervical extension, for all severities of discogenic spondylosis when there was abnormal motion, intersegmental hypomobility was predominant. Also of note was the presence of paradoxical motion, which occurred in 11% of the intervertebral motion units without discogenic spondylosis [usually at the C7-T1 intervertebral motion unit (86%)]. From the data it can be concluded that there are trends which occur with differing amounts of discogenic spondylosis when considering intersegmental cervical sagittal motion. However, additional detailed study is required to corroborate the findings and determine what their clinical significance is.


Asymmetry of the occipital condyles: a computer-assisted analysis.

To ascertain the incidence of asymmetry of the occipital condyles. The a priori hypothesis of symmetry (which is pervasive both in the determination of "subluxation" and in the means chosen for correction) was tested. Data set design was used with a convenience sample. The condyles were chosen for convenience of visualization from plain film radiographs. The study was retrospectively designed, albeit with prospective implications for analytic investigation. The research department of Cleveland Chiropractic College, Kansas City, MO. One hundred fifty-one submentovertex radiographs were examined using a computer-assisted digitizer. The radiographs were randomly obtained from the patient files of two doctors of chiropractic who routinely use this type of radiograph in their patient analysis. The study was a side-to-side comparison of the condyles, which were subject to neither intervention nor change. Surface area of left and right condyles for 151 examined pairs. Analysis with Pearson's correlation coefficient implied a lack of symmetry between condyles (r = .37; p < .0001). The scatterplot revealed values widely dispersed about the regression line, and the standard error of the estimate was 36.7. The data suggest that the underlying premise of symmetry in chiropractic analysis needs to be reexamined.


The immediate effect of manipulation versus mobilization on pain and range of motion in the cervical spine: a randomized controlled trial.

The main objective of this study is to compare the immediate results of manipulation to mobilization in neck pain patients. The patients were compared in a randomized controlled trial without long-term follow-up. The study was conducted at an outpatient teaching clinic on primary and referred patients. One hundred consecutive outpatients suffering from unilateral neck pain with referral into the trapezius muscle were studied. Fifty-two subjects were manipulated and 48 subjects were mobilized. The mean (SD) age was 34.5 (13.0) yr for the manipulated group and 37.7 (12.5) yr for the mobilized group. Sixteen subjects had neck pain for less than 1 wk, 34 subjects had pain for between 1 wk and 6 mo and 50 subjects had pain for more than 6 mo. Seventy-eight subjects had a past history of neck pain. Thirty-one subjects had been involved in an injurious motor vehicle accident and 28 subjects had other types of minor trauma to the neck. There were no significant differences between the two treatment groups with respect to history of neck pain or level of disability as measured by the Pain Disability Index. The patients received either a single rotational manipulation (high-velocity, low-amplitude thrust) or mobilization in the form of muscle energy technique. Prior to and immediately after the treatments, cervical spine range of motion was recorded in three planes, and pain intensity was rated on the 101-point numerical rating scale (NRS-101). Both pre- and post-test measurements were conducted in a blinded fashion. The results show that both treatments increase range of motion, but manipulation has a significantly greater effect on pain intensity. Eighty-five percent of the manipulated patients and 69% of the mobilized patients reported pain improvement immediately after treatment. However, the decrease in pain intensity was more than 1.5 times greater in the manipulated group (p = .05). This study demonstrates that a single manipulation is more effective than mobilization in decreasing pain in patients with mechanical neck pain. Both treatments increase range of motion in the neck to a similar degree. Further studies are required to determine any long-term benefits of manipulation for mechanical neck pain.


Leg length inequality.

The (Pacific) Consortium for Chiropractic Research has taken the initiative to research, investigate and provide information directed toward health care accountability, including evaluation of health care measures and practices. This article is a review of the literature pertaining to leg length inequality (LLI). Leg length measures are utilized by various health care providers as an indicator of biomechanical imbalance and necessity for care. Following the overview of the incidence, classifications and clinical significance, emphasis was placed on methods of evaluation, especially the visual "quick" leg length measurement procedure. Finally, a brief section on conservative care has been provided. The information and studies reviewed were obtained from Medline, the Index to Chiropractic Literature, Chiropractic Research Archives Collection, Physiotherapy Index, Chiropractic Literature Analysis and Retrieval System, various technique manuals, personal communication with technique research advisors, proceedings and dissertations. REFERENCE SELECTION: In spite of the widespread use of leg length measures, there is still much controversy associated with LLI, its biomechanical significance and the reliability of the various measurement systems employed. Therefore, the 182 references utilized were selected on the basis of applicable information, especially studies that provided a detailed methodological design with statistical analysis regarding evaluation procedures. There appears to be a lack of agreement concerning incidence, classification and point of clinical significance. However, the manifestations or consequences of LLI demonstrate greater accordance. Of the three most commonly utilized evaluation methods, radiographic measures such as the scanograms are recognized as the most reliable procedure for the evaluation of anatomical LLI. Much controversy exists with some of the clinical orthopedic methods and the visual "quick" leg check. Because there is such a vast range in estimates of reliability, few if any definitive conclusions can be made regarding these methods. Given this, it is evident that more research is needed before the use of certain orthopedic and visual checks are considered reliable and valid.


Short lever, specific contact articular chiropractic technique.

To identify what has been theorized and/or written about the manual techniques generically classified as direct, but using specific contacts on short levers while using forces that may vary in speed and amplitude. A search of the MEDLINE bibliographic database using MeSH key words (chiropractic/methods; osteopathic manipulation) was conducted. The Index to Chiropractic Literature and Chiropractic Research Abstracts Collection were searched through the past 10 yr using the key terms of chiropractic-methods; chiropractic techniques; manipulation, chiropractic; manipulation, osteopathic, manipulation, spinal; and manipulation, joint. A hand search of text-books was undertaken as well as review of the references included in books, monographs and collected papers. Studies in English were included, but it must be noted that these include publications that are not peer reviewed or refereed in any way. Descriptions that discussed aspects of manipulative procedures with the appropriate characteristics were extracted by a single author. Very little was found or accessible using traditional methods of literature retrieval. Lack of common terminology as well as multiple technique systems developed in isolation contributed to the problem. Descriptions of characteristics for the attributes of techniques which use specific contacts on short levers are provided. The high-velocity, low-amplitude thrust technique is presented as one of the oldest and most widely used forms of manual medicine and remains one of the most frequently used forms of manual medicine. Derivation of applied forces as well as speculation as to the roll of specificity are discussed. This type of review is considered the first step in the evaluative process for a chiropractic technique procedure. It demonstrates that very little has been written in an accessible fashion relative to techniques which use specific contacts on short levers while applying forces that may vary in speed and amplitude. Statements made concerning the appropriateness or effectiveness consist of opinions only, with no reference to any form of injury or testing. The need for clinical trials and studies comparing different techniques is great.


Treatment of Bell's palsy by mechanical force, manually assisted chiropractic adjusting and high-voltage electrotherapy.

Bell's palsy is a relatively common, painful, unilateral facial paralysis of unknown etiology. While often claimed to be successfully treated by chiropractic methods, no standard treatment approach is generally recognized within the profession, nor has any report of chiropractic treatment of this condition been reported in the literature. This article discusses two cases of Bell's palsy successfully treated by mechanical force, manually assisted chiropractic adjusting technique combined with high-voltage electrotherapy. While these two cases do not necessarily represent any rule for the chiropractic treatment of Bell's palsy by the methods used, or for other chiropractic methods that may be used, they do represent an initial time/treatment "baseline" by which future inter- and intratechnique comparisons may be made for the determination of relative effectiveness.


Magnetic resonance imaging of the wrist: occult osseous lesions.

This article illustrates the magnetic resonance imaging (MRI) features of occult osseous lesions in three different patients. All three patients suffered from a history of wrist trauma, but had negative plain film radiographs. The three types of occult lesions (bone bruise, microfracture and chronic osseous damage) are described, and their MRI characteristics are demonstrated by proton density and T2 weighted images. MRI proved to be the diagnostic modality of choice for the detection of posttraumatic intrinsic marrow changes.


Distinct signals in human immunodeficiency virus type 1 Pr55 necessary for RNA binding and particle formation.

The human immunodeficiency virus type 1 (HIV-1) gag gene product Pr55 self-assembles to form virus-like particles when expressed in Spodoptera frugiperda cells using recombinant baculoviruses. The particles resemble immature HIV and are released from the infected cell into the culture medium. Using this system we have progressively truncated the gag open reading frame from the C terminus and examined each deleted gag protein for its particle-producing capability. We show that deletion of Pr6 and deletions that progressively remove the distal region of the Pr7 domain, including one Cys-His box thought to function as an RNA capture signal, do not affect particle formation. However deletion of two Cys-His boxes causes production of slightly larger particles with altered sedimentation properties. Sequence-specific North-Western assays using an RNA probe representative of the HIV-1 packaging signal revealed specific RNA binding by all mutants that maintained both Cys-His boxes. However, deletion of one Cys-His box reduced RNA binding substantially and loss of two Cys-His boxes abolished binding entirely. We conclude that HIV-1 gag particle formation per se does not require viral RNA encapsidation, but that it may act as a cofactor in the condensation of the immature core. Further deletion of gag sequences upstream of the Cys-His boxes led to the abolition of particle-forming ability, and we show that one boundary of the gag sequence necessary for particle formation lies within eight amino acids spanning one of the known protease cleavage sites at the C terminus of Pr24.


Strain-specific selection of genome segments in avian reovirus coinfections.

To determine whether selection of genome segments in coinfections is strain-specific, chicken embryo fibroblasts were coinfected with avian reovirus strain 883 and one of three other avian reovirus strains (176, S1133 and 81-5). Viral progeny from each coinfection (883 x 176, 883 x S1133 or 883 x 81-5) was serially passaged at a low m.o.i. The electropherotypes of the coinfection progeny and those of the plaque-derived clones obtained from passages 1 and 20 were analysed. Two 883 segments (M2 and S2) were found to be selected in the 883 x 176 coinfection, three 883 segments (M2, M3 and S2) in the 883 x S1133 coinfection, and only one 883 segment (M3) in the 883 x 81-5 coinfection, i.e. different 883 genome segments were selected in the three coinfections. It was, therefore, concluded that selection of genome segments in a coinfection of a given cell line is virus strain-specific. The selection of genome segments in coinfections was shown to be due to enhanced infectivity of the reassortants that were formed in the coinfections. In addition, defective interfering particles that lack the S1 segment were identified in the 883 x 81-5 coinfection progeny following serial passage. Selection of genome segment(s) in coinfections as described herein may have potential importance on the effect and production of divalent or multivalent vaccines.


Nucleotides 9 to 11 of the influenza A virion RNA promoter are crucial for activity in vitro.

The 12 nucleotide conserved sequence at the 3' end of influenza A virion RNA is sufficient to function as a promoter in vitro. By introducing point mutations in all 12 positions of this promoter in model RNA templates and studying the efficiency of RNA synthesis in vitro, we show that only three nucleotides, residues 9, 10 and 11, are crucial for activity, although other nucleotides play a significant but less important role. Additions or deletions within the promoter are tolerated, resulting in either an increase or a decrease in promoter activity, depending on the mutation introduced; in some cases premature termination is caused. Taking these observations into account, a model for RNA polymerase binding and copying of the promoter is discussed.


Activation of protein kinase C inhibits adenovirus VA gene transcription in vitro.

We report here that activation of protein kinase C (PKC) results in the inhibition of adenovirus virus-associated (VA) gene transcription in vitro. The involvement of PKC in this inhibition is supported by the fact that the addition of PKC inhibitors to transcription reactions in which the PKC cofactor phosphatidyl serine (PS) was present resulted in increased levels of transcription compared to those in reactions in which PKC activity was stimulated in the absence of PKC inhibitors. Furthermore, based on these in vitro studies we propose that the inhibition of VA gene transcription is possibly due to the inability of the VA gene to form an active transcription complex following the activation of PKC. This conclusion was drawn from in vitro data which demonstrated that PS stimulation of endogenous PKC present in cell extracts resulted in failure to isolate a fully initiated, stable transcription complex.


Molecular basis of hepatitis B virus serotype variations within the four major subtypes.

Amino acid residues 101 to 180 of hepatitis B surface antigen (HBsAg) were predicted by sequencing the corresponding part of the S gene of hepatitis B virus (HBV) DNA in 46 HBsAg-positive sera, which had been subtyped by immunodiffusion with respect to d/y, w/r, w1 to w4 and q. The sequences of the nine different HBV serotypes defined by these specificities were found to be homogeneous proving that they represent consistent variations of HBV at the genomic level. Residue 127 was found to be important as were Pro, Thr and Leu for w1/w2, w3 and w4, respectively. Five residues were found to differ between ayw1 and ayw2. These were at positions 134 (Phe instead of Tyr), 143 (Thr instead of Ser), 159 (Ala instead of Gly), 161 (Tyr instead of Phe) and 168 (Val instead of Ala). However, all these residues were shared by ayw1 and adw2, implying that Arg122 was also important for w1 expression. All genomes expressing r, apart from one ayr strain, had an Ile126, which might explain the pseudo-allelism of w1 to w4 in relation to r, since this substitution might influence the w epitope. There were two regions where adw4q- and adrq- differed from all the q+ subtypes. These were located at residues 158 and 159, and at residues 177 and 178, where both the q- subtypes had amino acid substitutions in adjacent positions. The mapping of the epitopes defining these antigenic specificities will help to link information on the world-wide distribution of HBsAg subtypes to future molecular epidemiology with regard to HBV.


Phenotypic characterization of three temperature-sensitive mutations in the vaccinia virus early gene transcription initiation factor.

Vaccinia virus gene D6R encodes the small subunit of the virion early gene transcription initiation factor. Three temperature-sensitive mutations have been mapped to this gene. The biochemical phenotype exhibited by each mutation was examined. All mutants displayed altered viral protein synthesis in pulse-labelling analyses at both the permissive and non-permissive temperatures. The onset of early protein synthesis was delayed, and the rate of early protein synthesis was reduced in each case. Furthermore the shut-off of both host and early protein synthesis was delayed. In pulse-chase experiments, the stability of the D6R protein in E93- or C46-infected cells was shown to be reduced at 40 degrees C relative to that at 31 degrees C. Early mRNA was quantified in cells at 2 h post-infection and shown to be reduced substantially. The ability of each mutant virus to support transcription in vitro was examined at both temperatures and, of the three mutants, only S4 transcription was shown to exhibit reversible temperature sensitivity.


Characterization of the nucleotide sequence of the Lymantria dispar nuclear polyhedrosis virus DNA polymerase gene region.

The DNA polymerase gene of the Lymantria dispar multinucleocapsid nuclear polyhedrosis virus (LdMNPV) was cloned and sequenced. The predicted DNA polymerase protein (1113 amino acids, 115.9K) was found to have an amino acid identity of 48% with the corresponding gene of the Autographa californica MNPV (AcMNPV). It contains five domains associated with substrate binding, primase interaction, and pyrophosphate hydrolysis and three domains associated with 3'-->5' exonuclease activity common to other DNA polymerases. A region with a conserved TATA promoter and a CAGT mRNA start site sequence motif was identified and shown to be transcribed by RNA polymerase II, indicating that the LdMNPV DNA polymerase gene is expressed as an early gene. An open reading frame possibly expressed as a late gene, oriented in the opposite direction and overlapping the N-terminal coding region of the DNA polymerase gene was found in the LdMNPV sequence and was shown to be conserved in the same position in AcMNPV.


Equilibrium and kinetic analysis of Autographa californica nuclear polyhedrosis virus attachment to different insect cell lines.

The kinetic and equilibrium attachment of Autographa californica nuclear polyhedrosis virus (AcMNPV) to seven insect cell lines was evaluated. Kinetic experiments revealed differences of up to 10-fold in the infection rates among cell lines. Equilibrium binding also varied between cell lines and was saturable. The Tn 5B1-4 and Tn F cell lines had the highest virus binding affinities and infection rates and exhibited diffusion-limited attachment. The rate of infection appears to be limited by the rate of attachment. For the Tn 5B1-4 cells the physical to infective particle ratio for AcMNPV was 5.3. From the Scatchard analyses, the cell lines Tn 5B1-4 and Tn F displayed affinities of 2.35 x 10(10) M-1 and 1.60 x 10(10) M-1, respectively, with 6000 and 13,700 binding sites per cell. The insect cell line Hz 1075, which is not susceptible to AcMNPV infection, displayed a much lower, but saturable, binding of AcMNPV with 900 sites/cell and an affinity of 1.1 x 10(10) M-1. Unlabelled AcMNPV, but not Lymantria dispar MNPV could compete with labelled AcMNPV for binding sites. There were 93 to 96% reductions in virus cell binding following pretreatments of cells with three proteases, suggesting the involvement of a cellular protein component in virus binding. Tunicamycin, an inhibitor of N-linked glycosylation and expression of some membrane proteins on the cell surface, reduced virus binding in a dose-dependent manner suggesting a role for glycoprotein(s) in binding. However there was no evidence for the direct involvement of oligosaccharides in attachment. Metabolic inhibitors of oligosaccharide trimming and competition binding assays using simple sugars caused no measurable reductions in virus binding. These findings suggest that AcMNPV attachment to insect cells is receptor-mediated via a glycoprotein component(s); the direct involvement of oligosaccharide moieties in binding is unlikely.


Protein synthesis in pupae of the silkworm Bombyx mori after infection with nuclear polyhedrosis virus: resistance to viral infection acquired during pupal period.

Protein synthesis has been studied in pupae of the silkworm Bombyx mori (Bm) infected with nuclear polyhedrosis virus (BmNPV) at various stages of the pupal period. Nascent proteins were labelled by injection of [35S]methionine into pupae and then analysed by SDS-PAGE. Temporal regulation of synthesis of infected cell-specific proteins (ICSPs) in pupae was demonstrated by electrophoretic analysis of the proteins labelled at different times post-infection (p.i.). The rate of ICSP synthesis reached a maximum at 4 to 5 days p.i., exceeding the rate of synthesis of cellular proteins in uninfected pupae by about twofold. The viral proteins p10 and polyhedrin were the most abundant products synthesized late in the infection. Both proteins were found to be associated with the nuclear matrix after fractionation of nuclei from infected pupae. Two virus-induced phosphoproteins, pp35 and ppB, were found to be the major acceptors of labelled phosphate from [gamma-32P]ATP during in vitro phosphorylation of proteins in pupal homogenates, nuclei and nuclear extracts. These proteins had electrophoretic mobilities comparable to those of structural phosphoproteins of BmNPV virions with M(r)s of 35K and 11K to 16K, respectively. The latter polypeptide was identified as the major DNA-binding protein of the virus. The susceptibility of silkworms to BmNPV decreased markedly during the pupal period. Following injection of BmNPV all young pupae acquired polyhedrosis and finally died whereas most of the older pupae did not exhibit disease and completed metamorphosis normally. Moreover, the later in the pupal period the silkworms were infected, the lower the production of polyhedrin in diseased pupae.


The nucleotide sequence of parsnip yellow fleck virus: a plant picorna-like virus.

The complete sequence of 9871 nucleotides (nts) of parsnip yellow fleck virus (PYFV; isolate P-121) was determined from cDNA clones and by direct sequencing of viral RNA. The RNA contains a large open reading frame between nts 279 and 9362 which encodes a polyprotein of 3027 amino acids with a calculated M(r) of 336212 (336K). A PYFV polyclonal antiserum reacted with the proteins expressed from phage carrying cDNA clones from the 5' half of the PYFV genome. Comparison of the polyprotein sequence of PYFV with other viral polyprotein sequences reveals similarities to the putative NTP-binding and RNA polymerase domains of cowpea mosaic comovirus, tomato black ring nepovirus and several animal picornaviruses. The 3' untranslated region of PYFV RNA is 509 nts long and does not have a poly(A) tail. The 3'-terminal 121 nts may form a stem-loop structure which resembles that formed in the genomic RNA of mosquito-borne flaviviruses.


The nucleotide sequence of RNA-1 of raspberry bushy dwarf virus.

Raspberry bushy dwarf virus (RBDV) has isometric, 33 nm diameter particles and a bipartite RNA genome. Sequencing of the larger component (RNA-1) showed that it consists of 5449 nucleotides and contains one large open reading frame encoding a putative translation product with a calculated M(r) of 190,000. Comparisons of this polypeptide with non-structural proteins of other plant viruses revealed significant homologies with those of alfalfa mosaic virus (AlMV), brome mosaic virus (BMV), cucumber mosaic virus (CMV) and tobacco mosaic virus (TMV). Thus RBDV belongs to the supergroup of 'Sindbis-like' plant viruses. The translation product of RBDV RNA-1 contains motifs characteristic of proteins with polymerase, methyltransferase and helicase activities, suggesting that this protein is involved in the replication of the viral RNA. Thus in RBDV, as in TMV, all three functional domains are combined in the single protein, whereas in AlMV, BMV and CMV these domains are distributed over the proteins encoded by RNA-1 and RNA-2. These findings support the idea that RBDV should be placed in a distinct virus genus for which the name idaeovirus has been proposed.


Resistance to phloem transport of potato leafroll virus in potato plants.

A 'double-graft sandwich' technique in which sections of potato stem from different potato cultivars were grafted between a susceptible healthy stock plant and a potato leafroll virus (PLRV)-infected scion was used to study the rate of phloem transport of PLRV in cultivars differing in resistance to PLRV infection (IR) and accumulation (AR). Resistance to phloem transport (i.e. delayed PLRV systemic movement) was found in Bismark cultivar (IR A(S)). This was independent of IR and AR as the rate of movement in Bismark cultivar was markedly slower than that in Omega and Spunta (IR AR), Delaware (I(S) AR), and Desiree and Renova (I(S) A(S)) cultivars. It operated in Bismark cultivar stems of two different ages, but did not operate against potato virus X (PVX) and was not influenced by previous infection with this virus. Aphid vector (Myzus persicae) feeding preferences and colonization rates differed between cultivars, but the cultivar characteristics responsible were unrelated to IR, AR or resistance to phloem transport. Delayed systemic movement of PLRV out of leaves inoculated with viruliferous aphids was independent of AR and resistance to phloem transport, and remained unaffected by previous infection with PVX. It was also independent of cultivar factors causing different aphid feeding preferences and colonization rates, but may be linked to IR.


The nucleotide sequence of tomato mottle virus, a new geminivirus isolated from tomatoes in Florida.

A new geminivirus, tomato mottle virus (TMoV), affecting tomato production in Florida has been cloned and sequenced. Sequence analysis of the cloned replicative forms of TMoV revealed four potential coding regions for the A component [2601 nucleotides (nt)] and two for the B component (2541 nt). Comparisons of the nucleotide sequence of the TMoV genome with those of other whitefly-transmitted geminiviruses indicate that TMoV is a typical bipartite geminivirus of the New World and is closely related to but distinct from abutilon mosaic virus.


Induction of a novel protein kinase in pupae of the silkworm Bombyx mori after infection with nuclear polyhedrosis virus.

Protein kinases induced by Bombyx mori nuclear polyhedrosis virus in pupae of the silkworm B. mori were examined by activity gel analysis using phosvitin as a protein substrate. The method involved PAGE of the soluble fraction from pupae under native conditions and in the presence of SDS, followed by in situ renaturation of proteins and recovery of protein kinase activity in the intact gel. A novel protein kinase able to phosphorylate phosvitin was detected in the infected pupae from 2 days post-infection. This enzyme was not present in uninfected silkworms at any stage of the pupal period. The novel kinase activity was found by SDS-PAGE to be associated with a single polypeptide with an apparent M(r) of 50K. However, on electrophoresis under native conditions its activity was associated with a set of polypeptides with similar but not identical electrophoretic mobilities. Microheterogeneity of the catalytically active polypeptides suggests that the virus-induced protein kinase undergoes post-translational modification during the course of infection.


Comparison of the thymidine kinase genes from three entomopoxviruses.

The entomopoxviruses (insect poxviruses) of eastern spruce budworm (Choristoneura fumiferana), two year cycle spruce budworm (C. biennis) and the Indian red army worm (Amsacta moorei) are being studied in our laboratory for their potential as biological insecticides and expression vectors. These viruses characteristically replicate in the cytoplasm of insect cells and produce occlusion bodies that serve to protect the virion from the environment. By analogy to mammalian poxviruses, they should also contain a viral thymidine kinase (TK) that functions in viral DNA synthesis. The replication of the A. moorei entomopoxvirus was inhibited by bromodeoxyuridine whereas the baculovirus of Autographa californica was insensitive to this drug. This result was a biochemical indication that entomopoxviruses contained a kinase that phosphorylated this nucleoside analogue and thus viral DNA synthesis was inhibited. TK genes from the three different insect poxviruses were identified, cloned and sequenced. The sequences of the TK genes of the entomopoxviruses were closely related and exhibited 63.2% identity and 9.9% similarity at the protein level. However, there was only 36.7% identity and 13.6% similarity when these enzymes were compared to their mammalian poxvirus counterpart in vaccinia virus. Finally, one entomopoxvirus TK gene was expressed in Escherichia coli mutants lacking the enzyme. These bacteria were converted to a phenotype that could incorporate radioactive thymidine into their chromosomal DNA. The results presented in this paper provide impetus for the design of a recombinant entomopoxvirus expression system in which foreign genes could be introduced into the viral TK locus under selective pressure from bromodeoxyuridine.


A myxoma virus intergenic transient dominant selection vector.

The purpose of this study was to construct an intergenic transfer vector which can be used for the generation of recombinant myxoma viruses (MVs) expressing a foreign gene insert. Recombinant MVs expressing the Escherichia coli lacZ gene were constructed in vitro by transfection of MV-infected rabbit cells with a transfer expression vector, and isolated under growth conditions selecting for transient expression of the E. coli gpt gene. The effect of inserting foreign DNA sequences between the viral thymidine kinase gene and open reading frame MF8a upon the transcription of these genes was investigated.


Human immunodeficiency virus type 1-infected HL-60 cells are capable of both monocytic and granulocytic differentiation.

We have used the human myelomonocytic cell line HL-60 as a model system to determine whether human immunodeficiency virus type 1 (HIV-1) infection affects differentiation of myeloid progenitor cells. HL-60 cells were infected with three HIV-1 isolates (IIIB, NL4-3 and PM213). HIV-1 antigen expression and cytopathicity in HL-60 cells infected with each of the three isolates was delayed by approximately 15 days as compared to those in the prototypic T cell line, H9. Chronically infected HL-60 cells and clonal lines derived from them were treated with dimethyl formamide (DMF) and induced to differentiate into granulocytes. Approximately the same percentage of these cells as of DMF-treated, uninfected HL-60 cells differentiated. Superoxide production by infected and uninfected DMF-induced cells was similar. Likewise, approximately the same percentage of cells in infected and uninfected cultures became adherent and were positive for non-specific esterase when monocytic differentiation was induced. The data demonstrate that HL-60 cells infected with HIV-1 are capable of morphological and functional granulocytic and monocytic differentiation.


Murine gammaherpesvirus 68 establishes a latent infection in mouse B lymphocytes in vivo.

Murine gammaherpesvirus 68 (MHV-68) is able to persist in spleen cells of infected mice. To determine the cell type harbouring persistent virus, spleen cells from infected animals were separated into immunoglobulin (Ig)-positive (B cell-enriched), Ig-negative (T cell-enriched) and plastic-adherent (macrophage-enriched) fractions. These cells were co-cultivated with permissive BHK-21 cells in an infectious centre assay. The consistent recovery and enrichment of infectious centres in the Ig-positive fraction clearly demonstrates that B cells are a major site of virus persistence/latency. This observation indicates that MHV-68 is biologically similar to Epstein-Barr virus and other members of the B cell lymphotropic gammaherpesvirus 1 subgroup.


Sequence analysis of bovine adenovirus type 3 early region 3 and fibre protein genes.

The DNA sequences of the early region 3 (E3) and fibre protein genes of bovine adenovirus type 3 (BAd3) have been determined and the amino acid sequences predicted to be encoded by their open reading frames (ORFs) compared to those of the fibre and E3 proteins from other Ads. One of the BAd3-E3 proteins contains a region homologous to the 14.7K E3 protein of human Ad5 (HAd5). The putative BAd3 fibre protein contains a number of regions homologous to the HAd2 fibre protein sequence, but is predicted to be 244 amino acids longer owing to an increase in the number of repeating structural motifs of hydrophobic amino acid residues in the shaft region. Sequences to the left of the BAd3-E3 gene region contained the 3' end of another ORF with extensive identity with the hexon-associated protein precursor (pVIII) of HAd2. Like mouse Ad1 and canine Ad1, the BAd3 E3 gene is approximately 1.5 kb, about half the size of the E3 region of HAd2 and HAd5.


Molecular evidence for the origin of the widespread Venezuelan equine encephalitis epizootic of 1969 to 1972.

Venezuelan equine encephalitis (VEE) virus is a mosquito-borne pathogen that has caused encephalitis in equine species and humans during sporadic outbreaks in the western hemisphere. The last, and most widespread, VEE outbreak occurred in South America, Central America, Mexico and the U.S.A. (Texas) during 1969 to 1972. We have cloned and sequenced the genome of a virulent VEE subtype I-AB virus, strain 71-180, isolated in Texas in 1971. Thirty-four nucleotide differences were detected between the genome of 71-180 virus and that of the subtype I-AB Trinidad donkey (TRD) virus isolated during the 1943 VEE epizootic in Trinidad. Fifteen nucleotide changes occurred in the non-structural genes, 16 in the structural genes and three in the 3' non-coding region. Only six of the nucleotide differences resulted in amino acid substitutions: one change in each of non-structural proteins nsP1 and nsP3, two in the E2 envelope glycoprotein, one in the 6K polypeptide and one in the E1 envelope glycoprotein. The close genetic relationship between 71-180 virus and TRD virus, commonly used for production of formalin-inactivated VEE vaccines, suggests that incompletely inactivated virulent vaccine virus may have been the source of this and other VEE outbreaks. Use of formalized virulent virus was discontinued during the 1969 to 1972 panzootic. No VEE epizootics have been reported since the introduction of the live attenuated TC-83 vaccine virus.


In vitro culture for the detection of infectious human parvovirus B19 and B19-specific antibodies using foetal haematopoietic precursor cells.

The inability to culture human parvovirus B19 in standard cell lines has rendered investigation of clinical samples for the presence of infectious virus problematic. Using haematopoietic precursors derived from first trimester foetal liver as targets for infection, and non-isotopic in situ hybridization to detect intracellular viral DNA, we have assessed infectivity in stored serum samples taken from nine volunteers at different stages following intranasal inoculation with parvovirus B19. Infectious virus was detected as early as 3 days after inoculation, the cessation of infectivity correlating with the rise in specific IgM. In all but two samples, infectivity correlated with the detection of B19 DNA by dot-blot hybridization, although in vitro culture was 10-fold more sensitive than dot-blot hybridization. B19 DNA was detected by the polymerase chain reaction in serum from one volunteer up to 36 days after inoculation, although samples containing specific antibody were non-infectious. Infection of erythroid precursors was completely inhibited by preincubation of virus with serum containing high titre B19-specific IgM and IgG. Unexpectedly, this was associated with a strong B19 DNA hybridization signal within the cytoplasm of phagocytic macrophages. This culture and detection system is a rapid and sensitive means of detecting infectious virus in serum samples, and of assessing the neutralizing ability of B19-specific antibodies.


Binding of influenza A virus NS1 protein to dsRNA in vitro.

The non-structural protein NS1 of influenza A virus exhibits two modes of RNA-binding activity. One is sequence-specific binding to minus-sense virus RNA with either a 5'- or 3'-terminal common sequence as reported previously. The other was identified as binding to dsRNA and this activity did not show sequence specificity. The affinity of binding to dsRNA was much higher than that to ssRNA. A short miniature virion RNA forming a panhandle structure by pairing between the 5'- and 3'-terminal common sequences bound NS1 with higher affinity and stability than did a dsRNA of similar sequence and length.


War zone traumas and posttraumatic stress disorder symptomatology.

The diagnosis and clinical understanding of posttraumatic stress disorder (PTSD) rests upon the explicit identification of traumatic experiences that give rise to a well-defined constellation of symptoms. Most efforts to investigate the characteristics of these experiences have attempted to specify war zone stressors as objectively as possible. In this study, we add specification of the psychological meaning of war zone stressors to their objective specification. Eleven traumas are organized in terms of four roles that veterans played in the initiation of death and injury; namely, target, observer, agent, and failure. These roles can be ordered in terms of the degree of personal responsibility involved in the initiation of death and injury. The relationships of these roles to current symptomatology were examined in combination with a set of objective measures of war zone stressors. The sample consisted of the first 1709 Vietnam theater veterans who were assessed in a national evaluation of the PTSD Clinical Teams initiative of the Department of Veterans Affairs. Results show that having been a target of others' attempts to kill or injure is related more uniquely than any other role to symptoms that are diagnostic criteria for PTSD. On the other hand, having been an agent of killing and having been a failure at preventing death and injury are related more strongly than other roles to general psychiatric distress and suicide attempts.(ABSTRACT TRUNCATED AT 250 WORDS)


Chronic posttraumatic stress disorder and diagnostic comorbidity in a disaster sample.

Research has indicated significant comorbid psychopathology with chronic posttraumatic stress disorder (PTSD) in samples of war veterans. The present paper examines the issue of comorbidity in a disaster sample to learn whether findings from veterans generalized to this event. A total of 193 subjects exposed to the Buffalo Creek dam collapse of 1972 were examined 14 years later using diagnoses derived from the Structured Clinical Interview for DSM-III (SCID). Past and present PTSD was found in a significant portion of the sample. Major depression was the next most common diagnosis and was highly related to PTSD. Anxiety disorders were also common. The overlap with other diagnoses was quite similar to that found in a sample of Vietnam veterans we studied earlier, except that the disaster sample had fewer dysthymic disorders, substance abusers, and antisocial personality disorders. Possible explanations for comorbidity in chronic PTSD were discussed and it was suggested that the morphology of PTSD may be quite stable in at least some other nonveteran trauma populations.


Malignant memories. Reluctance to utilize mental health services after a disaster.

This report describes the reluctance of individuals exposed to a man-made disaster to utilize formal mental health services. Measures were obtained in an initial screening 6 months after a shooting for 24 exposed school personnel. Data from the initial screening were compared for those who did not participate in a follow-up screening 12 months later (N = 11) and those who did (N = 13). Follow-up nonparticipants reported: more posttraumatic stress disorder symptoms, especially avoidance; recall of life threat during the event; feeling depressed; and an increase in positivity toward victims but not about their work or mental health professionals. The authors conclude that some individuals may avoid formal mental health services because they serve as cues for malignant memory retrieval and discuss implications for service delivery.


Persecutory delusions and the self-serving bias. Evidence from a contingency judgment task.

The self-serving attributional bias was studied in matched groups of patients with persecutory delusions, patients with major affective disorder, and normal controls, (N = 14 in each group). On a preprogrammed computer task, subjects mainly won points in one condition and mainly lost points in the other. Subjects were asked to estimate the degree of control they thought they had over winning or losing in the two conditions. In comparison with the normal subjects and the psychiatric controls, the deluded subjects showed a greater self-serving bias, as evidenced by their perceived greater control over outcomes in the win condition. These findings are interpreted as consistent with the hypothesis that persecutory delusions function as a defense against low self-esteem.


History of the discovery of neuronal death in embryos.

The German anatomists, M. Ernst and A. Glücksmann, deserve credit for the discovery of widespread cell death in embryonic tissues, including the nervous tissue. In 1934, V. Hamburger described a significant hypoplasia in dorsal root ganglia (DGR) and lateral motor columns, following the extirpation of limb buds in chick embryos. In the early 1940s, Dr. Rita Levi-Montalcini in Turin (Italy) repeated the experiment and suggested that the hypoplasia might result from the death of young differentiated neurons. In a joint reinvestigation, published in 1949, large numbers of degenerating neurons were described in brachial DRG, following wing bud extirpations. In the same embryos, Dr. Levi-Montalcini observed massive neuronal death in cervical and thoracic DRG which had not been affected by the operation. This was the discovery of naturally occurring neuronal death. Long after the discovery of Nerve Growth Factor (NGF) it was recognized that NGF and natural neuronal death are two sides of the same coin: the latter results from an insufficient supply of the former by the target tissues.


The role of target size in neuronal survival.

A loss of about half of the trochlear motor neurons occurs during the course of normal development in duck and quail embryos. The role of the size of the target muscle in controlling the number of surviving motor neurons was examined by making motor neurons innervate targets either larger or smaller in size than their normal target. In one experiment the smaller trochlear motor neuron pool of the quail embryo was forced to innervate the larger superior oblique muscle of the duck embryo. This was accomplished by grafting the midbrain of a quail embryo in the place of the midbrain of a duck embryo. Results indicated that no additional quail trochlear motor neurons were rescued in spite of a considerable increase in target size. In another experiment the larger trochlear motor neuron pool of the duck embryo was made to innervate the smaller superior oblique muscle of the quail embryo. This resulted in loss of some additional neurons; however, the number of surviving motor neurons was not proportionate to the reduction in target size. These experiments failed to provide support for the hypothesis that the size of the target muscle controls the number of surviving motor neurons. Although contact with target is necessary for survival of neurons, factors other than the number or size of target cells are involved in the control of motor neuron numbers during development.


The relationship of intramuscular nerve branching and synaptogenesis to motoneuron survival.

The target has been considered for some time to play a major role in allowing neurons to survive the period of naturally occurring cell death. For the motoneurons that innervate the chick limb, evidence is presented that suggests access to target-derived trophic factor via intramuscular nerve branches and synapses may be important in regulating neuronal survival. Alterations in branching and synapse formation produced by activity blockade as well as by alteration of adhesion molecule function are shown to result in changes in motoneuron survival consistent with the proposed hypothesis. The relevance of these observations to the numerical-matching hypothesis of vertebrate neuronal cell death is also considered.


Neuron death in the developing avian isthmo-optic nucleus, and its relation to the establishment of functional circuitry.

The present review covers all the published data on neuron death in the developing avian isthmo-optic nucleus (ION), which provides a particularly convenient situation for studying the causes and consequences of neuron death in the development of the vertebrate central nervous system. The main conclusions are as follows: The naturally occurring neuron death in the ION is related both temporally and causally to the ION's formation of afferent and efferent connections. The ION neurons need to obtain both anterograde and retrograde survival signals in order to survive during a critical period in embryogenesis. They may compete, at least for the retrograde signals, but the nature of the competition is still unclear. The retrograde signals are modified by action potentials. Neurons dying from a lack of anterograde survival signals can be distinguished morphologically from ones dying from a lack of retrograde signals. The neuron death refines circuitry by selectively eliminating neurons with "aberrant" axons projecting to the "wrong" (i.e., ipsilateral) retina or to the "wrong" (topographically inappropriate) part of the contralateral retina.


Cell death and the creation of regional differences in neuronal numbers.

Regional variations in cell death are ubiquitous in the nervous system. In the retina, cell death in retinal ganglion cells is elevated in the retinal periphery and may be important in setting up the initial conditions that produce central retinal specializations such as an area centralis or visual streak. In central visual system structures, pronounced spatial and spatiotemporal inhomogeneities in cell death are seen both in layers and regions of the lateral geniculate nucleus and superior colliculus; similar indications of inhomogeneities are seen in those nonvisual structures that have been examined. Cell death in the cortex is highly nonuniform, by layer and by cortical area. A variety of possible functions for these regional losses are proposed, in the context of a uniform mechanism for cell death that allows it to assume multiple functions.


Matters of life and death in the songbird forebrain.

Male zebra finches learn a specific vocal pattern during a restricted period of development. They produce that song in stereotyped form throughout adulthood, and are unable to learn new song patterns. Development of the neural substrate for song learning and behavior is delayed relative to other brain regions, and neural song-control circuits undergo dramatic changes during the period of vocal learning due to both loss of neurons as well as incorporation of newly generated neurons. In contrast, canaries do learn new song patterns in adulthood and modify their vocal repertoires each breeding season. Adult canaries also maintain a large population of dividing cells in the ependymal zone of the telencephalon, and vast numbers of newly generated neurons migrate out to become incorporated into functional circuits and replace older neurons. We review the relationships between cellular and behavioral aspects of song learning in both zebra finches and canaries, as well as the role of gonadal hormones in regulating diverse aspects of the song-control system.


Regulation of motoneuron death in the spinal nucleus of the bulbocavernosus.

A sexual dimorphism in the number of motoneurons in the spinal nucleus of the bulbocavernosus (SNB) of rats is engendered by a sex difference in ontogenetic cell death. Testicular secretions, specifically androgenic steroids, reduce SNB motoneuron death in males. The fate of the target muscles generally mirrors that of the motoneurons, and androgens appear to exert their effects upon the target muscles, sparing the motoneurons as a secondary consequence. Treatment with ciliary neurotrophic factor can also spare SNB motoneurons in newborn females, raising the possibility that this factor normally mediates androgen's effect upon motoneuron survival. The ontogeny of calcitonin gene-related peptide immunoreactivity is delayed in SNB cells compared with other motoneurons and is further delayed in the SNB cells of females. In both sexes, calcitonin gene-related peptide is detected after the period of SNB motoneuron death is complete. A sex difference in motoneuron number is also seen in the human homologue of the SNB and, because ontogenetic death of motoneurons in humans overlaps the period of androgen secretion, may arise in a manner similar to that in the rat SNB.


Cell death in the oligodendrocyte lineage.

We have recently found that about 50% of newly formed oligodendrocytes normally die in the developing rat optic nerve. When purified oligodendrocytes or their precursors are cultured in the absence of serum or added signalling molecules, they die rapidly with the characteristics of programmed cell death. This death is prevented either by the addition of medium conditioned by cultures of their normal neighboring cells in the developing optic nerve, or by the addition of platelet-derived growth factor (PDGF) or insulin-like growth factors (IGFs). Increasing PDGF in the developing optic nerve decreases normal oligodendrocyte death by up to 90% and doubles the number of oligodendrocytes, suggesting that this normally occurring glial cell death might result from a competition for limiting amounts of survival signals. These results suggest that competition for limiting amounts of survival factors is not confined to developing neurons, and raise the possibility that a similar mechanism may be responsible for some naturally occurring cell deaths in nonneural tissues.


Mechanisms of neuronal death in brain aging and Alzheimer's disease: role of endocrine-mediated calcium dyshomeostasis.

This paper reviews evidence that brain aging and Alzheimer's disease (AD) are somehow closely related and that the hippocampus (CA1) is highly vulnerable to cell loss under both conditions. In addition, two current lines of evidence on the mechanisms of hippocampal cell loss with aging are considered, including studies of neuronal calcium dysregulation and studies of cumulative glucocorticoid (GC) neurotoxicity. Moreover, recent electrophysiological studies have shown that excess glucocorticoid activation of hippocampal neurons increases the influx of calcium through voltage-activated calcium channels. Second messenger systems may mediate the steroid modulation of calcium channels. Therefore, it is hypothesized that excess glucocorticoid activation and neuronal calcium dysregulation may be two phases of a single process that increases the susceptibility of neurons to neurodegeneration during aging and Alzheimer's disease.


Programmed neuronal death in insect development.

Programmed death in the developing nervous system of insects serves to remove obsolete neurons, generate segmental specializations and sexual dimorphism, as well as adjust neuronal number. This diversity is also reflected in the mechanisms which control the death of these neurons. In general, but not without exception, these deaths occur independent of target fate, while endocrine cues, segmental identity, and neural signalling often play critical roles. In addition, the programmed death of at least some neurons can be delayed by behavioral feedback. The study of neuronal death in Drosophila and the cloning of an ecdysteroid receptor bring the promise of understanding the genetic factors and molecular events that regulate this phenomenon.


Insect muscle as a model for programmed cell death.

Programmed cell death (PCD) is a fundamental component of development in virtually all animals. Despite the ubiquity of this phenomenon, little is known about what tells a cell to die, and less still about the physiological and molecular mechanisms that bring about death. One system that has proven to be very amenable for the study of PCD is the intersegmental muscle (ISM) of the tobacco hawkmoth Manduca sexta. These giant muscle cells are used during the eclosion (emergence) behavior of the adult moth, and then die during the subsequent 30 h. This review uses the ISMs as a model system to address questions that are basic to any cell death system, including the following: (1) how do cells know when to die; (2) what physiological changes accompany death; (3) what are the molecular mechanisms that mediate death; and (4) do all cells die by the same process? For the ISMs, the trigger for PCD is a decline in the circulating titer of the insect molting hormone, 20-hydroxyecdysone (20-HE). During cell death there are rapid decreases in both the myofibrillar sensitivity to intracellular calcium and the resulting force of fiber contraction. The ability of the ISMs to undergo PCD requires the repression and activation of specific genes. Two of the repressed genes encode actin and myosin. One of the upregulated presumptive cell-death genes encodes polyubiquitin, which appears to play a critical role in the rapid proteolysis that accompanies ISM death.(ABSTRACT TRUNCATED AT 250 WORDS)


Molecular genetics of cell death in the nematode Caenorhabditis elegans.

In C. elegans, cell death can be readily studied at the cellular, genetic, and molecular levels. Two types of death have been characterized in this nematode: (1) programmed cell death, which occurs as a normal component in development; and (2) pathological cell death, which occurs aberrantly as a consequence of mutation. Analysis of mutations that disrupt programmed cell death in various ways has defined a genetic pathway for programmed cell death which includes genes that perform such functions as the determination of which cells die, the execution of cell death, the engulfment of cell corpses, and the digestion of DNA from dead cells. Molecular analysis is providing insight into the nature of the molecules that function in these aspects of programmed cell death. Characterization of some genes that mutate to induce abnormal cell death has defined a novel gene family called degenerins that encode putative membrane proteins. Dominant alleles of at least two degenerin genes, mec-4 and deg-1, can cause cellular swelling and late onset neurodegeneration of specific groups of cells.


Typical absence seizures in adults: clinical, EEG, video-EEG findings and diagnostic/syndromic considerations.

Eighteen women and five men had typical absences. These included 10% of a consecutive hospital series of 200 adult patients with epileptic disorders. The absences began between the ages of seven and 46 years and varied in type and severity. Twenty patients also had generalised tonic-clonic seizures, ranging in frequency from one in a lifetime to one per month. Myoclonic jerks of the limbs occurred in 11 patients but were not associated with the absence attacks. Eyelid myoclonus consistently occurred with absence attacks in four patients and perioral myoclonus in two patients. Absence status occurred in five patients. Absence seizures were frequently unrecognised or misdiagnosed as complex partial seizures. Satisfactory control was achieved with sodium valproate. Electroencephalography, particularly video-electroencephalography, was invaluable in the diagnosis, but focal abnormalities in seven patients might have been erroneously interpreted as indicating partial seizures. This series showed that clinical and EEG manifestations are often syndrome-related and that there are more epileptic syndromes with typical absences than those presently recognised.