Comparative antitumor effects of hormonal ablation, estrogen agonist, estrogen cytotoxic derivative, and antiestrogen in the PAIII rat prostatic adenocarcinoma.

The effects of hormonal ablation, estrogen, estrogen-derived cytotoxic agent, and estrogen antagonist therapies used clinically were evaluated on in vitro colony formation, in vivo growth, and lymphatic and pulmonary metastasis of the PAIII tumor. Ventral prostatic and seminal vesicle weights were evaluated in the same animals to assess androgen-related responses. Estradiol, estramustine phosphate, and testosterone had no effects on PAIII colony formation in vitro. Castration, hypophysectomy, estradiol benzoate, and estramustine phosphate treatment of PAIII-bearing Lobund Wistar rats produced significant (P less than 0.05) regression of male accessory sex organs. Of these treatments, only hypophysectomy had significant (P less than 0.05) inhibitory effects on primary PAIII growth and lymphatic and pulmonary metastasis. LY117018 [6-hydroxy-2-(p-hydroxyphenyl)benzo(b)thien-3-yl-p-2-(l-pyrrolidin yl)ethoxy phenyl ketone] has antiestrogenic activity but produces no significant agonist responses. LY117018 had no effect upon PAIII colony formation in vitro. Following s.c. implantation of PAIII cells, LY117018 (2.0, 10.0, or 20.0 mg/kg s.c.) had no effect on primary tumor growth in the tail. In vitro LY117018 administration produced marked antimetastatic effects. In a dose-dependent manner, LY117018 inhibited PAIII metastasis to the gluteal (97%) and iliac lymph nodes (88%) (P less than 0.05 for both). LY117018 also maximally inhibited pulmonary metastasis by 86% (P less than 0.05). Maximal regression of 42% for ventral prostatic and 35% for seminal vesicle weights were also seen after LY117018 administration (P less than 0.05 for both). Co-administration of estradiol benzoate had no antagonistic effect upon the antitumor responses produced by LY117018. The mechanism of action of LY117018 is not known. The failure of estradiol benzoate to affect PAIII growth and metastasis supports the contention that the responses to LY117018 are not attributable to simple antagonism of estrogen action. LY117018 may be exerting its antitumor effects through autocrine, paracrine, or endocrine mechanisms. LY117018 represents a class of agents with potential utility in treating metastatic cancer of the prostate.     

Increased feeding and neuropeptide Y (NPY) but not NPY mRNA levels in the hypothalamus of the rat following central administration of the serotonin synthesis inhibitorp-chlorophenylalanine

Neurons containing serotonin (5-HT), a potent anorexic agent, come into contact with neuropeptide Y-ergic neurons, that project from the arcuate nucleus (ARC) to the paraventricular nucleus (PVN). NPY powerfully stimulates feeding and induces obesity when injected repeatedly into the PVN. We hypothesize that 5-HT tonically inhibits the ARC-PVN neurons and that balance between the two systems determines feeding and energy homeostasis. This study aimed to determine whether central injection of the 5-HT synthesis inhibitor p-chlorophenylalanine (pCPA), which increases feeding, increased hypothalamic NPY and NPY mRNA levels. pCPA (10 mg/kg in 3 μl) was administered into the third ventricle either as a single injection (n = 8) or daily for 7 days (n = 8). Control rats received a similar injection of saline. pCPA significantly increased food intake compared with controls after both single and repeated injections (P < 0.05). NPY levels were measured by radioimmunoassay in microdissected hypothalamic extracts. NPY levels in the acutely treated group were significantly increased in the paraventricular nucleus (PVN; by 41%,P = 0.01), anterior hypothalamic area (AHA; by 34%,P < 0.01) and lateral hypothalamic area (LHA; by 41%,P < 0.02). In the 7-day-treated group, NPY levels were also increased in the same areas, i.e. PVN (by 24%,P < 0.01), AHA (by 30%,P < 0.01) and LHA (by 38%,P = 0.01). There were no significant changes in the ARC or any other region or in hypothalamic NPY mRNA levels. pCPA administration increased NPY levels in several regions notably the PVN. This is a major site of NPY release, where NPY injection induces feeding. We suggest that the hyperphagia induced by pCPA is mediated by increased NPY levels and secretion in the PVN. This is further evidence for interactions between NPY and 5-HT in the control of energy homeostasis.     

Molecular Cloning and Characterization of the Genes Coding for the Highly Immunogenic Cluster of 90-Kilodalton Envelope Proteins from the Chlamydia psittaci Subtype That Causes Abortion in Sheep

Proteins present in the outer membrane of chlamydiae that are involved in mucosal epithelial cell infection must clearly be identified and characterized if we are to understand and modify the pathogenic mechanisms utilized by these organisms. We have identified and isolated a family of four genes encoding putative outer membrane proteins (POMPs), a group of proteins of approximately 90 kDa present in the outer membrane of the subtype of Chlamydia psittaci that causes ovine enzootic abortion (strain S26/3). These proteins, although minor components, are major immunogens, as shown by the immunoblotting of chlamydial outer membrane complexes with postabortion sheep sera, and are therefore potential diagnostic and/or protective antigen candidates. Immunoblotting of the expressed amino- and carboxy-terminal halves of one of the POMPs with postabortion sheep sera showed that the major humoral immune response appeared to be directed solely against the amino-terminal half. This result, in combination with the positive immunofluorescence staining of S26/3-infected cells using POMP-specific (specific to the amino-terminal half of the proteins) monoclonal antibodies, suggests the probable surface localization of the POMPs and, more specifically, the surface exposure of the amino-terminal half of these proteins. The four pomp genes are highly homologous, sharing 82 to 100% similarity with each other (two of the genes are identical). Genes with strong and weak homologies were also detected in C. psittaci avian and feline pneumonitis strains, respectively. No pomp homologs were found in strains of C. trachomatis and C. pneumoniae, but this does not preclude their existence. The absence of homology with various subtypes of C. pecorum, which complicate the diagnosis of the ovine abortion subtype, indicates the possible suitability of the these 90-kDa proteins as serodiagnostic antigens.     

An Immunohistochemical Study of the Pathology of Fatal Malaria: Evidence for Widespread Endothelial Activation and a Potential Role for Intercellular Adhesion Molecule-1 in Cerebral Sequestration

The sequestration of parasitized erythrocytes in the microvasculature of vital organs is central to the pathogenesis of severe Plasmodium falciparum malaria. This process is mediated by specific interactions between parasite adherence ligands and host receptors on vascular endothelium such as intercellular adhesion molecule-1 (ICAM-1) and CD36. Using immunohistochemistry we have examined the distribution of putative sequestration receptors in different organs from fatal cases of P.falciparum malaria and noninfected controls. Receptor expression and parasite sequestration in the brain were quantified and correlated. Fatal malaria was associated with widespread induction of endothelial activation markers, with significantly higher levels of ICAM-1 and E-selectin expression on vessels in the brain. In contrast, cerebral endothelial CD36 and thrombospondin staining were sparse, with no evidence for increased expression in malaria. There was highly significant co-localization of sequestration with the expression of ICAM-1, CD36, and E-selectin in cerebral vessels but no cellular inflammatory response. These results suggest that these receptors have a role in sequestration in vivo and indicate that systemic endothelial activation is a feature of fatal malaria.     

Genetic linkage analysis identifies new proximal and distal flanking markers for the X-linked agammaglobulinemia gene locus, refining its localization in Xq22

Genetic linkage analysis has been instrumental in mapping thegene for X-linked agammaglobulinemia (XLA) to the proximal longarm of the human X chromosome, to Xq22. Due to the relativerarity of this disease the localization of the gene within Xq22has remained imprecise. We have investigated twenty-nine familiesaffected by XLA and have found no recombinants with the DXS178locus in over 30 informative meioses. DXS178 is now the mostreliable and informative locus for use in pre-natal diagnosisand carrier detection of XLA. In addition, we have identifiednew closely linked proximal and distal flanking markers forXLA, DXS442 and DXS101, respectively. These loci are separatedby 2cM, considerably reducing the extent of DNA within whichthe XLA locus can be contained. This will open up the way formore directed positional cloning efforts for the isolation ofthe XLA gene.     

Serum alpha 2-macroglobulin levels in diabetes.

Serum alpha 2-macroglobulin levels have been determined in diabetic patients by quantitative radial immunodiffusion and compared with those observed in age- and sex-matched controls. In addition, the results in diabetics have been analysed with respect to such variables as the age and sex of the patient, the duration of disease, treatment, control, and the occurrence of retinopathy or nephropathy. The alpha 2-macroglobulin levels in diabetic patients were found to be significantly higher than in age- and sex-matched controls, thus confirming previous observations. However, these differences were most apparent in the more extreme age groups. Multiple regression analysis also revealed that the only variables contributing significantly to the regression apart from age and sex were control and retinopathy.     

Activity-dependent control of bulk endocytosis by protein dephosphorylation in central nerve terminals

Bulk endocytosis is the process by which nerve terminals retrieve large amounts of synaptic vesicle membrane during periods of strong stimulation intensity. The process is rapidly activated and is most probably calcium dependent in a similar manner to synaptic vesicle exocytosis. This article briefly summarizes the current knowledge of bulk endocytosis with respect to its activation, kinetics and molecular mechanism. It also presents recent data from our laboratory showing that the dephosphorylation of a group of endocytosis proteins called the dephosphins by the Ca²⁺-dependent protein phosphatase calcineurin is key to the activity-dependent stimulation of the process. Possible downstream effectors of calcineurin are discussed such as the large GTPase dynamin I and its phosphorylation-dependent interaction partner syndapin I.     

Complex CGH alterations on chromosome arm 8p at candidate tumor suppressor gene loci in breast cancer cell lines

Loss of genetic material from chromosome arm 8p occurs frequently in human breast carcinomas, consistent with this region of the genome harboring one or more tumor suppressor genes (TSGs). We used the complementary techniques of microsatellite-based LOH, high-density FISH, and conventional CGH on 6 breast cancer cell lines (MCF7, SKBR3, T47D, MDA MB453, BT549, and BT474) to investigate the molecular cytogenetic changes occurring on chromosome 8 during tumorigenesis, with particular emphasis on 6 potential TSGs on 8p. We identified multiple alterations of chromosome 8, including partial or complete deletion of 8p or 8q, duplication of 8q, and isochromosome 8q. The detailed FISH analysis showed several complex rearrangements of 8p with differing breakpoints of varying proximity to the genes of interest. High rates of LOH were observed at markers adjacent to or within PCM1, DUSP4/MKP2, NKX3A, and DLC1, supporting their status as candidate TSGs. Due to the complex ploidy status of these cell lines, relative loss of 8p material detected by CGH did not always correlate with microsatellite-based LOH results. These results extend our understanding of the mechanisms accompanying the dysregulation of candidate tumor suppressor loci on chromosome arm 8p, and identify appropriate cellular systems for further investigation of their biological properties.