GRP78-targeting subtilase cytotoxin sensitizes cancer cells to photodynamic therapy.

Glucose-regulated protein 78 (GRP78) is an endoplasmic reticulum (ER)-resident chaperone and a major regulator of the unfolded protein response (UPR). Accumulating evidence indicate that GRP78 is overexpressed in many cancer cell lines, and contributes to the invasion and metastasis in many human tumors. Besides, GRP78 upregulation is detected in response to different ER stress-inducing anticancer therapies, including photodynamic therapy (PDT). This study demonstrates that GRP78 mRNA and protein levels are elevated in response to PDT in various cancer cell lines. Stable overexpression of GRP78 confers resistance to PDT substantiating its cytoprotective role. Moreover, GRP78-targeting subtilase cytotoxin catalytic subunit fused with epidermal growth factor (EGF-SubA) sensitizes various cancer cells to Photofrin-mediated PDT. The combination treatment is cytotoxic to apoptosis-competent SW-900 lung cancer cells, as well as to Bax-deficient and apoptosis-resistant DU-145 prostate cancer cells. In these cells, PDT and EGF-SubA cytotoxin induce protein kinase R-like ER kinase and inositol-requiring enzyme 1 branches of UPR and also increase the level of C/EBP (CCAAT/enhancer-binding protein) homologous protein, an ER stress-associated apoptosis-promoting transcription factor. Although some apoptotic events such as disruption of mitochondrial membrane and caspase activation are detected after PDT, there is no phosphatidylserine plasma membrane externalization or DNA fragmentation, suggesting that in DU-145 cells the late apoptotic events are missing. Moreover, in SW-900 cells, EGF-SubA cytotoxin potentiates PDT-mediated cell death but attenuates PDT-induced apoptosis. In addition, the cell death cannot be reversed by caspase inhibitor z-VAD, confirming that apoptosis is not a major cell death mode triggered by the combination therapy. Moreover, no typical features of necrotic or autophagic cell death are recognized. Instead, an extensive cellular vacuolation of ER origin is observed. Altogether, these findings indicate that PDT and GRP78-targeting cytotoxin treatment can efficiently kill cancer cells independent on their apoptotic competence and triggers an atypical, non-apoptotic cell death.

Cellular phenotypes and spatio-temporal patterns of lymphatic vessel development in embryonic mouse hearts.

BACKGROUND: The origin of cardiac lymphatics from venous endothelial cells or from scattered lymphangioblasts has been discussed in the literature. We aimed to establish the stage when lymphatic vessels appear in the developing mouse heart, the location of the first lymphatics, and to define cellular phenotypes of growing lymphatics. RESULTS: We found that scattered Lyve-1-positive cells located in the subepicardial area of developing heart expressed CD45, CD68, F4/80, or CD11b but not CD31. Prox-1(+)/Lyve-1(+) cellular cords or vessels were found to invade 12.5-13.5-dpc hearts via two routes: from the venous pole, i.e., dorsal atrioventricular sulcus, or on the dorsal atrial surface from mediastinum and from the arterial pole, i.e., along the great arteries. The Prox-1(+)/Lyve-1(+) vessels were located among the Prox-1(+)/Lyve-1(-) cords and among the scattered Prox-1(-)/Lyve-1(+) cells. The Prox-1(+)/Lyve-1(-) cellular cords/tubules dominate initially at the arterial pole whereas Lyve-1(+)/Prox-1(-) cellular cords/tubules dominate initially on the venous pole, i.e., dorsal atrioventricular sulcus. The Lyve-1(+)/CD45(+), Lyve-1(+)/CD11b(+), Lyve-1(+)/F4/80(+) and Lyve-1(+)/CD68(+) cells were subsequently found to be co-opted to the wall of the developing lymphatic vessels while gaining Flk-1. CONCLUSIONS: Lymphatic primordia exhibit different cellular phenotypes and different spatiotemporal pattern on the venous pole as compared with the arterial pole of the heart.

Antitumor activity of bacteriophages in murine experimental cancer models caused possibly by inhibition of beta3 integrin signaling pathway.

Bacteriophages (phages) as bacterial viruses are generally believed to have no intrinsic tropism for mammalian cells. In this study the interactions between phages and various eukaryotic cells were investigated. Binding of phages to the membranes of cancer and normal blood cells was observed. Moreover, it was shown that the wild-type phage T4 (wtT4) and its substrain HAP1 with enhanced affinity for melanoma cells inhibit markedly and significantly experimental lung metastasis of murine B16 melanoma cells by 47% and 80%, respectively. A possible molecular mechanism of these effects, namely a specific interaction between the Lys-Gly-Asp motif of the phage protein 24 and beta3-integrin receptors on target cells is proposed. It was also shown that anti-beta3 antibodies and synthetic peptides mimicking natural beta3 ligands inhibit the phage binding to cancer cells. This is in line with the well-described beta3 integrin-dependent mechanism of tumor metastasis. It is concluded that the blocking of beta3 integrins by phage preparations results in a significant decrease in tumor invasiveness.

Current research on rapid cycling bipolar disorder and its treatment.

Rapid cycling is a pattern of presentation of bipolar disorder that specifies the course of the illness and is associated with a greater morbidity. The validity of rapid cycling as a distinct course modifier for bipolar disorder has been demonstrated and the term has been incorporated into the DSM-IV. The phenomenon of rapid cycling tends to appear late in the course of the disorder, occurs more frequently among females, and is more frequently seen in patients with bipolar type II disorder. Stimulants such as cocaine may also play some role in rapid-cycling. It is generally accepted that a recent history of rapid cycling predicts non-response to monotherapy with lithium and probably carbamazepine as well; however it is also possible that concurrent use of antidepressants may play a role in destabilizing the illness course under these agents. Thus, clinical considerations suggest that discontinuing antidepressants may facilitate the recovery process. Among clinically available monotherapies, valproate and lamotrigine appear to be the most useful clinically. However, other treatments such as lithium, carbamazepine, the atypical antipsychotic agents, thyroid hormone, and bupropion are frequently needed augmentation strategies. Electroconvulsive therapy may also prove efficacious in selected cases. The present paper provides a critical review of the evidence for the foregoing clinical issues in rapid cycling.

Clinical studies on the use of lamotrigine in bipolar disorder.

New mood stabilizers that possess efficacy in the depressed phase of bipolar disorder are needed. The use of marketed antidepressants puts bipolar patients at some increased risk for drug-induced hypomania/mania and rapid cycling. During the development of the antiepileptic, lamotrigine, the drug was observed to improve mood, alertness, and social interactions in some patients with epilepsy. These early observations provided the rationale for investigations into lamotrigine's potential efficacy in bipolar disorder. There are now 14 open clinical reports involving a total of 207 lamotrigine-treated patients with bipolar disorder that suggest this drug possesses a broad spectrum of efficacy in the management of the depressed, hypomanic, manic, and mixed phases of bipolar disorder. In an attempt to replicate and extend these preliminary open-label prospective findings, a series of multicenter, double-blind, placebo-controlled studies evaluating the efficacy and dose-response relationships of lamotrigine in the various phases of the illness, including both acute and maintenance designs in both bipolar I and II disorder, is ongoing.

Predictors of response to mood stabilizers.

Although lithium remains the preferred treatment of bipolar disorder, only 60 to 80% of patients with the classic presentation have an adequate response to this drug. When the response rate to lithium is considered across the entire spectrum of bipolar disorders, this rate probably decreases to 50%. Natural history, illness subtype, and comorbidity are all important general predictors of response to treatment. At present, the only predictors that seem to differentially favor divalproex, and possible, carbamazepine over lithium are mixed states and rapid cycling. An overview of clinical presentations that predict general and differential response to mood stabilizers is provided.

Co-product fiber digestibility: kinetic and in vivo assessment.

Nutrient digestion and kinetic characteristics of alfalfa hay, brewers grains, beet pulp, and cottonseed hulls were studied in two experiments. In the first experiment, these feedstuffs were fed with a mineral/vitamin supplement in a 4 x 4 Latin square to eight ewes to determine total tract digestibilities by 8-d total collection and rates of passage using Yb as a marker. In the second experiment samples of the same feedstuffs were incubated in situ in four ruminally fistulated steers in a 4 x 4 Latin square design to quantify ruminal disappearance of NDF and ADF. Three Dacron bags were placed in the rumen for each of six time periods: 6, 12, 24, 48, 72, and 96 h. Total tract digestibilities of DM ranged from 33% for cottonseed hulls to 78% for beet pulp. Digestibilities of NDF ranged from 32% for cottonseed hulls to 81% for beet pulp. Total tract retention times ranged from 74 h for beet pulp to 44 h for alfalfa. Retention time for alfalfa was lower (P < .05) than those for beet pulp or cottonseed hulls. Calculation of TDN values yielded values similar to those of NRC other than for cottonseed hulls, for which NRC values were 18 to 29% higher. Alfalfa hay and beet pulp were the feedstuffs with the most rapidly disappearing NDF, .124/h for alfalfa and .116/h for beet pulp, which were faster (P < .05) than those found in brewers grains and cottonseed hulls, .035 and .043/h, respectively. Potentially digestible NDF ranged from 55% for alfalfa hay to 94% for beet pulp.(ABSTRACT TRUNCATED AT 250 WORDS)

Ethanol-induced changes in Purkinje cells of the rat cerebellum. I. The ultrastructural changes following chronic ethanol intoxication (qualitative study).

The ultrastructural changes in the Purkinje cells from rat cerebellum following 3 months' ethanol treatment were investigated. The changes in both smooth (SER) and rough (RER) endoplasmic reticulum, mitochondria, microtubules lipofuscin granules of the Purkinje cells perikarya and of their dendrites were evaluated. The prominent dilatations were found in the hypolemmal cisternae as well as in SER canals. The canals in the lamellar bodies, which are a form of RER, were decreased in number, and sometimes they were disintegrated and dispersed throughout the cytoplasm of both perikarya and dendrites as single distended cisternae. The microtubules within dendrites were often disorderly oriented. The increased amount of lipofuscin granules in the Purkinje cells perikarya were also noticed. Our results suggest that chronic ethanol intoxication lead to disturbances in the synthetic capacity of the Purkinje cells, since the ultrastructure of both SER and RER was greatly changed.

Ethanol-induced changes in Purkinje cells of rat cerebellum. II. The ultrastructural changes after chronic ethanol intoxication. (Morphometric evaluation).

The morphometric approach to evaluation of some organelles of the rat cerebellar Purkinje cells after chronic ethanol intoxication according to the previously elaborated model (Jedrzejewska et al. 1990) was employed. In the experimental rats volumes of both the perikarya and nuclei in the Purkinje cells significantly increased. The quantitative, morphometric changes of both rough and smooth endoplasmic reticulum demonstrated, that in the perikarya and dendrites of the Purkinje cells the volume of rough endoplasmic reticulum decreased and the volume of smooth endoplasmic reticulum increased. The number of lipofuscin granules in the perikarya increased whereas the counts of microtubules per unit area in dendrites decreased. The statistical evaluation of parameters describing mitochondria and Golgi apparatus in ethanol-intoxicated animals did not show significant differences as compared with the control ones.

Signet-ring cells in squamous cell carcinoma of the cervix and in non-neoplastic ectocervical epithelium.

Two cases of cervical carcinoma and two cases of normal ectocervical epithelium with a squamous signet-ring cell component are presented. In one carcinoma the vacuolization was so prominent that it partially obliterated the epithelial pattern of the tumor. Paraffin specimens were studied with a panel of histo- and immunohistochemical stains and by electron microscopy. The vacuoles of the squamous signet-ring cells did not contain glycogen, mucopolysaccharides or masses of intermediate filaments. The findings are discussed in relation to possible human papillomavirus and Chlamydia trachomatis infection. The true nature and possible clinicopathologic implications of the squamous carcinoma with signet-ring cells remain to be established, but the phenomenon may cause diagnostic difficulties, especially in biopsy specimens.

Morphometric ultrastructural evaluation of secondary oocytes of mice receiving vincristine.

Secondary oocytes from mice receiving vincristine in amounts corresponding to therapeutic doses in human, in neoplasm treatment, were evaluated morphometrically and ultrastructurally. In the group of animals treated with vincristine an increase of the volume fraction of vesicular complexes and a decrease of the volume fraction of fibrous material were observed. It has also been demonstrated that, in secondary oocytes from mice receiving vincristine, the number of both multivesicular bodies and vesicles falling to one vesicular complex profile was higher than in the control group. The present results suggest that vincristine applied to females, even in therapeutic doses, may affect egg cells.

beta-Amyloid deposits within the cerebellum of persons older than 80 years of age.

The cerebellum, frontal cortex, hippocampal and parahippocampal regions of 100 patients older than 80 years, most of whom had died of stroke, were examined. Eighteen percent were diagnosed as clinically demented. On the specimens labeled previously with Thioflavin S and Bielschowsky method, immunohistochemical studies were performed with Fab (antigen-binding fragment) of the anti beta-amyloid antibody 4G8. Positive amyloid immunoreactivity was observed in the cerebrum in 71 of 100 cases, Cerebella of 31 subjects of 71 with cerebral amyloidosis also revealed amyloid deposits. They appeared in various morphological forms, such as diffuse plaques and focal subpial deposits, as well as classical and primitive neuritic plaques. Cases with amyloid in the cerebellum alone were not observed. Beta-amyloid deposits in the cerebellum were associated with a significant number of beta-amyloid plaques in the cerebrum, which showed other Alzheimer-type pathology, also in individuals without clinical symptoms of dementia. There was no correlation either between cerebellar amyloid deposits and clinical cerebellar symptoms or between the presence of diabetes mellitus, arterial hypertension, and neuropathological changes. A clear association of microglial cells with amyloid deposits in the cerebellum was demonstrated. In our experience, LN-1 and RCA-1 were not as suitable for formalin-fixed paraffin-embedded tissue, as was anti-ferritin. Negative staining for tau-1 and positive staining for anti-ubiquitin characterized neurites within primitive and classical plaques. No neurofibrillary pathology was detected in the cytoplasm of cerebellar neurons when we used anti tau-1 labeling.

Laminar analysis of the number of neurons, astrocytes, oligodendrocytes and microglia in the visual cortex (area 17) of 3-month-old rhesus monkeys fed a human infant soy-protein formula with or without taurine supplementation from birth.

The effect of taurine addition to a commercial (taurine-free) soy-protein formula on the development of the visual cortex has been examined in 3-month-old rhesus monkeys. The thickness, number of neurons, astrocytes, oligodendrocytes and microglia have been measured in the different laminae. There were no significant differences in cortical thickness, total number of neurons, astrocytes, oligodendrocytes, or microglia between the groups, but there were some significant differences in some laminae as well as some qualitative differences. The numerical density of neurons in layers IV-C alpha and V-2 was significantly greater in the taurine-supplemented monkeys than in those fed the taurine-free formula; the numerical density of astrocytes in layer IV-A was significantly greater and in layer IV-C alpha was significantly smaller in the taurine-supplemented monkeys than in those fed the taurine-free formula. A number of other measurements in the two groups approached significance. These differences indicate that there are developmental disadvantages in the visual cortex of rhesus monkeys raised on a taurine-free human infant formula in addition to those previously reported.

[The degradation of vicine and convicine in field bean meal by selected bacterial strains]. / Untersuchungen zum Abbau von Vicin und Convicin in Ackerbohnen mehl durch ausgewählte Bakterienstämme.

Vicine and convicine as natural occurring toxic compounds in Vicia faba are involved in the formation of hemolytic anemia (favism). Removal of these compounds needs high technological expense. Microbiological degradation with different strains of bacteria was tried. After 3 h incubation with Streptococcus faeciens convicine was converted completely and Vicine to about 95%.

Ultrastructural studies of the cells forming amyloid fibers in classical plaques.

Three-dimensional reconstruction and ultrastructural studies of classical plaques from the cortex of patients with Alzheimer's disease showed that microglial cells of the plaques are the amyloid-forming cells. The amyloid star of the single plaque represents the product of five or six microglial cells covering about 80% of the amyloid star surface. The amyloid fibers appear to be formed within altered cisterns of the endoplasmic reticulum. Distended cisterns form channels filled with amyloid fibers. Numerous vesicles derived from the Golgi apparatus appear to be attached to or fused with the amyloid-filled channels. Reconstruction of the amyloid star and the microglia cell pole that forms the amyloid star reveals three different zones of distribution of cytoplasmic organelles and amyloid deposits. The peripheral zone comprises channels filled with loosely packed amyloid fibers arranged in a parallel manner. The transient zone consists of a mixture of fusing amyloid channels and products of disintegration of cytoplasmic pockets, dense bodies and fragments of cellular membranes. The core of the amyloid star is composed of condensed, densely packed amyloid fibers that are free of cellular debris. Formation of the three zones supports the idea that the microglia/macrophages are not phagocytes but instead are the cells manufacturing the amyloid fibers.

Altered cartilage proteoglycans synthesized by chick limb bud chondrocytes cultured in serum-free defined medium.

Chick high-density culture chondrocytes synthesize cartilage-specific proteoglycans with much structural similarity to the proteoglycans made by cartilage in vivo. Such cultures can be maintained in a defined medium formulated in this laboratory in which chondrogenesis occurs without the addition of serum. The proteoglycans synthesized by the chondrocytes in the presence of defined medium are of a cartilage-specific structure but differ in some aspects from the proteoglycans made in serum-containing medium. While their buoyant density, ability to aggregate with hyaluronic acid, and keratan sulfate chain size are unchanged, the proteoglycans synthesized in defined medium have altered chondroitin sulfate chains. This chondroitin sulfate is of significantly larger size and has a different sulfation pattern relative to that produced in serum-containing medium. The larger size of the chondroitin sulfate results in a larger monomer size of the defined medium proteoglycans. These differences have implications about the regulation of the structure of chondroitin sulfate proteoglycans.