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Complexity and dynamics of HIV-1 quasispecies.

The human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins mediate virus entry into target cells by binding receptors of the cell membrane and fusing viral and cellular structures. In particular, recent crystallographic studies have clarified the complex role of the glycoprotein gp120 in the early phase of the infection. In this context the inter- and intra-host variability of the HIV-1 gp120 poses a major problem for the development of effective methods of immunization against this virus. In the present report, the relevant aspects emerging from the study of HIV-1 variability are addressed and several methodological approaches to evaluate HIV-1 diversity discussed.

Acquired Immunodeficiency Syndrome↗

The types of neurones in the neostriatum of the guinea pig (Cavia porcellus): Golgi and Klüver-Barrera studies.

The Golgi technique stain was used to reveal the cellular structure of the neostriatum (nucleus caudatus and putamen) in the guinea pig. The computerised reconstructions were made from Golgi impregnated neurones. On the basis of various criteria, 4 types of neurones were distinguished in the guinea pig neostriatum: 1. The rounded neurones (most numerous) with 5-8 thin dendritic trunks; 2. The triangular nerve cells with 3 thick dendritic trunks; 3. Two types of multipolar neurones differing in dendritic arborization pattern with 4-6 and 7-9 primary dendrites, respectively. 4. The pear-shaped cells, which divide into two distinctly different subpopulations.

Animals↗

Role of the cytoskeletal protein paxillin in oncogenesis.

The focal adhesion is an important cellular structure that is involved in cell signaling, cell motility, and oncogenic transformation. Paxillin is a unique adapter protein that is localized to the focal adhesion and is involved in regulating various functions of the focal adhesion. The predicted amino acid structure for paxillin shows at the amino-terminus five LD motifs, a proline-rich domain, several potential phosphorylation sites and four carboxy-terminal LIM domains. Paxillin interacts with cell surface receptors and the actin cytoskeleton and activates several signal transduction pathways that are known to regulate normal cell physiology. Because paxillin is a central protein within the focal adhesion, it is a common target of many different oncoproteins, such as BCR/ABL, v-Src, and E6. In this review we summarize the current knowledge about the structure/function of paxillin and its family members, its role in integrin, cytokine signaling, and oncogenic transformation.

Amino Acid Motifs↗

A subtilisin-like serine protease involved in the regulation of stomatal density and distribution in Arabidopsis thaliana.

Stomata are specialized cellular structures in the epidermis of aerial plant organs that control gas exchange (H(2)O release and CO(2) uptake) between leaves and the atmosphere by modulating the aperture of a pore flanked by two guard cells. Stomata are nonrandomly distributed, and their density is controlled by endogenous and environmental factors. To gain insight into the molecular mechanisms regulating stomatal distribution, Arabidopsis thaliana mutants with altered stomatal characteristics were isolated and examined. The sdd1-1 mutant exhibits a two- to fourfold increase of stomatal density and formation of clustered stomata (i.e., stomata that are not separated by intervening pavement cells), whereas the internal leaf architecture is not altered. The SDD1 gene was identified by map-based cloning. It encodes a subtilisin-like serine protease related to prokaryotic and eukaryotic proteins. We propose that SDD1 acts as a processing protease involved in the mediation of a signal that controls the development of cell lineages that lead to guard cell formation.

Amino Acid Sequence↗

Differentiation of a methylcholanthrene-induced sarcoma to a benign plexiform fibroneural tumor in an adult frog (Rana pipiens). Possible influence of host regenerative capacity.

A spindle cell sarcoma appeared 20 months after implantation of a pellet of 3-methylcholanthrene in the denervated foreleg of an adult frog, Rana pipiens. Its growth rate and cellular structure were observed over the subsequent 19 months, the former remaining constant for the first 14 months, then slackening markedly during the final 4 months. Serial biopsies disclosed maturation to a well-differentiated fibroneural tumor of benign appearance, the change taking place notably during the period of decelerated growth rate. An autotransplant of the tumor to the hind leg adopted the same growth pattern and maturation. Possible causes for this behavior are discussed.

Animals↗

Antioxidant enzyme expression and reactive oxygen species damage in prostatic intraepithelial neoplasia and cancer.

BACKGROUND: Oxidative stress results in damage to cellular structures and has been linked to many diseases, including cancer. The authors sought to determine whether the expression of three major antioxidant enzymes, copper-zinc superoxide dismutase (SOD1), manganese superoxide dismutase (SOD2), and catalase, was altered in human prostate carcinoma and its likely precursor, high grade prostatic intraepithelial neoplasia (PIN). The level of reactive oxygen species damage was evaluated by measuring the expression of the DNA adduct 8-hydroxydeoxyguanosine. METHODS: The authors evaluated the tissue expression of the antioxidant enzymes in prostate carcinoma by immunohistochemistry, immunogold electron microscopy, and enzymatic assay. The polymerase chain reaction was used to amplify and screen tissue specimens for the genes of SOD1, SOD2, and extracellular SOD (SOD3). Matched paraffin embedded tissue sections were evaluated by RNA in situ hybridization for expression of SOD1 and immunohistochemically for the DNA adduct 8-hydroxydeoxyguanosine. RESULTS: All prostatic tissues, including cancer, displayed immunoreactivity for the three antioxidant enzymes in epithelial cells, with no staining of the stroma, inflammatory cells, or endothelial cells. The number of immunoreactive cells was greater in benign epithelium than in PIN and cancer for each enzyme. The mean percentage and intensity of immunoreactive cells was greatest for SOD2, intermediate for SOD1, and lower for catalase. Staining in cancer was heterogeneous. Immunogold ultrasound studies revealed strong mitochondrial labeling for SOD2, which was greater in benign epithelium than in cancer; SOD1 labeling was invariably weaker, with nuclear labeling in benign epithelium and cytoplasmic labeling in cancer cells. There was no difference in enzyme activity for the three antioxidant enzymes between benign epithelium and cancer. No mutations were found in the 5 exons of SOD1, 5 exons of SOD2, and 3 exons of SOD3, except for 3 of 20 cases with polymorphisms for exon 3 of SOD1. Intense nuclear immunoreactivity for 8-hydroxydeoxyguanosine was present in fewer than 3% of epithelial cells, with no apparent differences among benign epithelium, PIN, and cancer. CONCLUSIONS: SOD1, SOD2, and catalase had lower expression in PIN and prostate carcinoma than in benign epithelium. The number of immunoreactive cells in PIN was similar to cancer, indicating that these are closely related. Enzyme activities were variable, with no difference between benign epithelial cells and cancer, although this lack of change in enzyme activity could have been due to the presence of contaminating benign cells within the cancer specimens. The results of reactive oxygen species damage were found only in the epithelium and not in the stroma. Expression of the DNA adduct 8-hydroxydeoxyguanosine was present in fewer than 3% of cells, with no apparent differences among benign epithelium, PIN, and cancer. These findings suggest that oxidative stress is an early event in carcinogenesis.

Aged↗

Probing antinuclear antibody specificities by peptide phage display libraries.

OBJECTIVE: To uncover the specificities of autoantibodies to nuclear proteins (ANA) in patients with juvenile rheumatoid arthritis (JRA). METHODS: Peptide ligands for ANA were selected by panning random peptide phage display libraries on antibodies binding to HEp-2 cells. Positive phage clones were identified by the immunoscreening technique. RESULTS: Groups of peptides were identified, some of which share the core motifs of KTTTnPY, RVADnL/I or RnNSPL. Perinuclear and nuclear staining of HEp-2 cells were obtained with patient serum antibodies binding to the phage displaying the core peptide motifs. In contrast, no significant reactivity was seen with the antibodies binding to the wild type phage. Antibodies to the phage displaying peptides containing some of the core motifs were detected more frequently in ANA-positive as compared to ANA-negative JRA patients. Homology search with the selected core motifs revealed a significant homology with a number of human nuclear proteins and proteins from potential infectious agents that could serve as trigger in the breakdown of tolerance. CONCLUSION: Panning of phage display libraries on antibodies reacting with cellular structures can lead to the identification of their specificities. Thus, the peptide epitopes reported here constitute additional information that may lead to the development of diagnostic tests and the identification of the parental antigens that initiated the B cell responses in patients with JRA.

Amino Acid Sequence↗

The DNA of annexin V-binding apoptotic cells is highly fragmented.

Jurkat leukemia cells induced to undergo apoptosis by treatment with an antibody against the Fas receptor have two annexin V (AV)-binding subpopulations: (a) single-positive cells that bind AV but not propidium iodide (PI); and (b) double-positive cells that bind AV and PI. The single-positive population is thought to represent an early stage of apoptosis. We have examined the relationship between AV binding and a classical characteristic of apoptosis, DNA fragmentation. Time course studies with Jurkat cells treated for 1, 2, or 4 h with anti-Fas indicated that the proportion of AV-binding cells was increased after 2 h. A significant increase in DNA fragmentation was observed only at 4 h as measured by the mean tail moment determined with the alkaline single cell gel electrophoresis (comet) assay. This correlation suggests a temporal relationship between the two parameters, but does not provide direct evidence of what happens in individual cells. We developed a method to measure fluorescent markers of cellular structure or function with a laser scanning cytometer and then perform the comet assay on the same cells. Cells in each AV-binding subpopulation were re-examined before and after electrophoresis. Most AV-/PI- cells had no DNA damage, although a few cells showed a pattern of damage characteristic for apoptosis. Double-positive cells all had damaged DNA; approximately half had the apoptotic pattern, and the rest had a pattern typical for necrosis. Nearly all of the single-positive cells had damaged DNA with the apoptotic pattern. Both AV-positive populations contained cells with little or no detectable DNA after electrophoresis, indicating that the DNA was highly fragmented. These results indicate that AV binding is an excellent marker for apoptotic cells, but that these cells already have fragmented DNA.

Annexin A5↗

[Development of corneal storage medium--first report. Examination of rabbit cornea].

PURPOSE: To develop and evaluate a new corneal storage medium with a simple formula by histological methods. METHODS: We compared two corneal storage media, containing minimum essential medium (MEM) and 2.5% chondroitin sulfate, pH 7.33 and osmolality of 320 mOsm/kg, to OPTISOL-GS. The differences in the two media were the molecular weight (MW) and source of chondroitin sulfate. MW of Medium I was 27,500 and MW of Medium II was 33,700. A cornea with scleral rim obtained from a Japanese white rabbit was stored in either Medium I or Medium II and the fellow cornea was stored in OPTISOL-GS for 7 or 14 days at 4 degrees C. Histological examination of corneal endothelial cells was performed both by scanning electron microscopy and by transmission electron microscopy. RESULTS: At day 7, there was no significant difference in histological findings among the rabbit corneas stored in OPTISOL-GS, Medium I, or Medium II. At day 14, corneas stored in OPTISOL-GS or Medium I showed similar histological findings. In Medium II, endothelial cells showed marked degeneration. CONCLUSION: The results of experiments with rabbit cornea indicated that OPTISOL-GS and Medium I could preserve endothelial cellular structure better than Medium II. The difference between Medium I and Medium II was only the MW of the chondroitin sulfate used. The MW may be an important factor to determine suitable chondroitin sulfate for developing a corneal storage medium.

Animals↗

Aggressive acid control: minimizing progression of Barrett's esophagus.

Barrett's esophagus (BE) is a change in the cellular structure of the lower esophagus believed by many to be caused by frequent acid exposure. The condition is troublesome because of a tendency toward cell proliferation, dysplastic transformation, and cancerous changes within the lesions. It is speculated that the pronounced increase in esophageal adenocarcinoma during the past 25 years may be attributable to acid reflux and associated BE. Strict acid control may cause lesion regression in these patients. However, pH monitoring to confirm adequate acid control is essential, and more than 1 drug may be needed to achieve adequate acid control and manage many patients. More research is needed to determine whether aggressive acid control reduces the dysplasia and cancer risk associated with BE.

2-Pyridinylmethylsulfinylbenzimidazoles↗

[Study of in vitro cultured microencapsulated adrenal cortical tissues of rats].

In order to investigate the growth of in vitro cultured microencapsulated adrenal cortical tissues of rats, we produced the microcapsules by using sodium alginate, calcium chloride and poly-L-lysine, etc. The adrenal glands were taken out of 24 rats, half of the adrenal glands were microencapsulated (capsule group) while the other half were left free (noncapsule group). After 36 hours' in vitro culture, the concentrations of aldosterone and cortisol in culture solution were measured by radioimmunoassay. The two groups continued to be cultured for 24 and 36 hours with ACTH stimulation. The concentrations of aldosterone and cortisol at 24 and 36 hours were detected respectively. Parts of adrenal cortical tissues were observed under light microscope and electron microscope. The results showed that the concentrations of aldosterone and cortisol in the microencapsulated adrenal cortical tissues were significantly higher than those of noncapsule group (P < 0.01). In the capsule group, the concentration of cortisol after 36 hours' ACTH stimulation was significantly higher than that before ACTH stimulation (P < 0.01). After 24 and 36 hours' ACTH stimulation, the concentrations of aldosterone and cortisol were significantly higher in the capsule group than in the noncapsule group. In the noncapsule group, regardless of ACTH stimulation, no significant change was seen in the concentration of aldosterone and cortisol (P > 0.05). The cellular structure of adrenal tissues remained intact under light microscopic and electron microscopic observations, and the cells remained alive well. These data suggest that in vitro cultured cells of microencapsulated adrenal cortical tissues remain alive well. Microcapsule has not influenced the secretory function of adrenal tissues. Microencapsulated tissues have a good response to ACTH stimulation.

Adrenal Cortex↗

Microbes, inflammation and atherosclerosis: will old pathology lessons guide new therapies?

Although attractive, the microbial pathogenesis theory for atherosclerosis remains unproven. Over the last century, microbiologists have invoked fulfillment of Koch's postulates to determine pathogen causality. Certainly a multifactorial disease process such as atherosclerosis unlikely will be due to a single microbial agent, an agent when transferred to another host, will always induce atherosclerosis. Conflicting epidemiological data also do not support a single causative agent. However, as presented here, considerable in vitro, animal, and human epidemiological data support the plausibility that infectious agents can promote a proinflammatory, procoagulant and proatherogenic environment in the vessel wall. Microbial genes and molecules can catalyze these processes and foil normal cellular events. But, must intact microbes enter the vessel wall or can microbial molecules incite immune responses from afar? A new focus on pathogen-induced auto-immunity toward vasculature has been presented. For example, microbes contain molecules that mimic host cellular components (55). An immune response to a pathogen may cross react with vessel wall cellular structures. This immune response enhanced by infection may lead to high levels of cross reacting auto-antibodies or auto-aggressive T-cells. Epstein has championed the concept of pathogen burden in support of this auto-immune theory (56). Individuals infected with multiple pathogens such as HSV-1, HSV-2, CMV, Helicobacter pylori, and Hepatitis A, have high C-reative protein levels (markers of inflammation) and the greatest relative risk for coronary artery disease (57). Thus, pathogens might contribute to the atherosclerotic process by promoting inflammatory responses. It is this author's view that microbes and inflammation do play a role in the pathogenesis of atherosclerosis (58). Infection may contribute to the process promoting vessel wall injury initiated by oxidized lipids, smoking derived oxidants, hypertensive shear or diabetes glyoxidized molecules. Inflammation and immune reactions in response to infection can exacerbate and act synergistically with all of the aforementioned vasculotoxic moieties. Continued investigations in the 21st century will determine if vaccines, antibiotics, anti-inflammatory agents or immunosuppressants will alter the picture the early 19th century pathologists observed under their monocular microscopes.

Arteriosclerosis↗

[Biochemical parameters predictive of neuronal damage in childhood].

INTRODUCTION: Hypoxemia, hypoxia and ischemia may induce deletereous effects on both metabolism and cellular structure, particularly at neuronal level. Early estimation of the potential severity of an acute cerebral hypoxic ischemic injury or other pathological conditions would be useful on making preventive or therapeutic decisions. On the basis of the physiopathological mechanisms involved in the brain damage related to hypoxic ischemic encephalopathy of the newborn, a number of metabolic parameters had been studied in the aim to provide an early and reliable marker of tissue injury for both diagnostic and prognostic purposes. OBJECTIVE: To provide a current revision about the diagnostic and prognostic value of various biochemical parameters determined in different body fluids and studied in the last years, the attention focusing in the hypoxic ischemic encephalopathy of the newborn. Design. For methodological purposes the exposition is structured in the following sections: 1. creatine kinase isoenzymes; 2. lactate; 3. lactate dehydrogenase, aspartate aminotransferase and hidroxybutirate dehydrogenase; 4. excitatory amino acids; 5. glial fibrillary acidic protein; 6. cytokines; 7. neuron specific enolase; 8. oxypurines; 9. cyclic adenosine monophosphate; 10. Others. CONCLUSIONS: The current role of the above mentioned biochemical parameters as predictors of brain damage and the future perspectives on this topic are discussed.

Aspartate Aminotransferases↗

[Primary pathological changes in cells infected with tick-borne encephalitis virus].

Synthesis and the content of RNA and protein in cells of the pig embryo kidneys infected with tick--borne encephalitis virus were studied by means of autoradiography and cytophotometry. Acceleration of synthesis and accumulation of RNA (in cellular structures) were estimated as over 125--200% of the initial level followed by a decrease in 12 hr when the virus release from the cells occurred. By the 2nd and 6--8th hr of infection (in the period of synthesis of early and late virus material), two peaks of protein contents over 123--131% were observed. The RNA and protein shifts are well correlated with the virus reproduction stages.

Animals↗

Circulating tumor cell clusters in the peripheral blood of colorectal cancer patients.

PURPOSE: Recently several reverse transcription-PCR techniques have been proven to be useful for the detection of circulating micrometastases. However, this way intact cell clusters that were found in animal experiments of prognostic value could not be detected. In this study, evaluation and modification of a commercial, cytokeratin-based, immunomagnetic cell separation method was performed for the detection of intact cell clusters in colorectal carcinoma patients. EXPERIMENTAL DESIGN: Thirty-two colon cancer patients (6 were in Dukes stage B, 13 in stage C, and 13 in stage D) and 20 healthy donor samples were evaluated. Immunomagnetic cell separation was performed from the buffy coat of peripheral blood samples (20 ml) using the Carcinoma Cell Enrichment Kit (Miltenyi Biotec, Bergisch Gladbach, Germany), avoiding any filtering steps. The enriched cell fraction was cytocentrifuged and immunocytochemically labeled using a pancytokeratin antibody (MNF116; Dako). RESULTS: Of 20 healthy samples, 2 contained one cytokeratin-positive cell. Of 32 single samples from malignant cases, 24 showed cytokeratin-positive cells. Tumor cell clusters, mixed-cell doublets (one cytokeratin-positive and -negative cell), and mixed-cell clusters were detected in 22 of 24 patients. In six cases, cytokeratin-positive dendritic-like cells were detected. Follow-up data indicate that chemotherapy cannot destroy all of the circulating tumor cell clusters. CONCLUSIONS: Using the methods presented, we could detect circulating colon cancer cells and cell clusters in colon carcinoma patients. Similar cellular structures were described previously only in rats. Present data prove that such structures are present in human colorectal cancer, too.

Cell Adhesion↗

[Phosphodiesterases of cyclic GMP].

Phosphodiesterases of cyclic nucleotides (PDEs) are enzymes hydrolyzing cGMP, cAMP or both and are regulated in several different ways. In this paper we summarize current data on structure, cellular and tissue localization, regulation and function of different PDE families that hydrolyze cGMP.

3',5'-Cyclic-AMP Phosphodiesterases↗

[The effect of 1, 25 (OH)(2)D(3)on growth and apoptosis in breast cancer cell line MCF-7].

OBJECTIVE: To study the effect of 1, 25-dihydroxyvitamin D(3) [1, 25 (OH)(2)D(3)] on growth and apoptosis in breast cancer cell line MCF-7. METHODS: We compared cell numbers by using MTT method, analyzed cell cycle and apoptosis percentage by flow cytometric, observed cellular structure and ultrastructure, determined quantitatively apoptosis cells by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), and detected expression of bcl-2 protein by Western Blot. RESULTS: Incubation with 1, 25 (OH)(2)D(3) 10(-7) mol/L, MCF-7 cells exhibited significant growth inhibition. Flow cytometric analysis indicated cell's G(0)/G(1) arrest along with increasing apoptotic peak and percentage. The action of cytotoxic drug Adr was strengthened. Western Blot showed that 1, 25 (OH)(2)D(3) could down-regulate bcl-2 protein expression. CONCLUSIONS: 1, 25 (OH)(2)D(3) could be a new hormone for the treatment of breast cancer.

Antineoplastic Agents, Hormonal↗