Inhibition of endoplasmic reticulum-associated degradation in CHO cells resistant to cholera toxin,Pseudomonas aeruginosa exotoxin A,and ricin

Many plant and bacterial toxins act upon cytosolic targets and must therefore penetrate a membrane barrier to function. One such class of toxins enters the cytosol after delivery to the endoplasmic reticulum (ER). These proteins, which include cholera toxin (CT), Pseudomonas aeruginosa exotoxin A (ETA), and ricin, move from the plasma membrane to the endosomes, pass through the Golgi apparatus, and travel to the ER. Translocation from the ER to the cytosol is hypothesized to involve the ER-associated degradation (ERAD) pathway. We developed a genetic strategy to assess the role of mammalian ERAD in toxin translocation. Populations of CHO cells were mutagenized and grown in the presence of two lethal toxins, ETA and ricin. Since these toxins bind to different surface receptors and attack distinct cytoplasmic targets, simultaneous acquisition of resistance to both would likely result from the disruption of a shared trafficking or translocation mechanism. Ten ETA- and ricin-resistant cell lines that displayed unselected resistance to CT and continued sensitivity to diphtheria toxin, which enters the cytosol directly from acidified endosomes, were screened for abnormalities in the processing of a known ERAD substrate, the Z form of alpha1-antitrypsin (alpha1AT-Z). Compared to the parental CHO cells, the rate of alpha1AT-Z degradation was decreased in two independent mutant cell lines. Both of these cell lines also exhibited, in comparison to the parental cells, decreased translocation and degradation of a recombinant CTA1 polypeptide. These findings demonstrated that decreased ERAD function was associated with increased cellular resistance to ER-translocating protein toxins in two independently derived mutant CHO cell lines.     

Intermittent preventive sulfadoxine-pyrimethamine treatment of primigravidae reduces levels of plasma immunoglobulin G, which protects against pregnancy-associated Plasmodium falciparum malaria

Pregnancy-associated malaria (PAM) is an important cause of maternal and neonatal suffering. It is caused by Plasmodium falciparum capable of inhabiting the placenta through expression of particular variant surface antigens (VSA) with affinity for proteoglycans such as chondroitin sulfate A. Protective immunity to PAM develops following exposure to parasites inhabiting the placenta, and primigravidae are therefore particularly susceptible to PAM. The adverse consequences of PAM in primigravidae are preventable by intermittent preventive treatment (IPTp), where women are given antimalarials at specified intervals during pregnancy, but this may interfere with acquisition of protective PAM immunity. We found that Kenyan primigravidae receiving sulfadoxine-pyrimethamine IPTp had significantly lower levels of immunoglobulin G (IgG) with specificity for the type of parasite-encoded VSA-called VSA(PAM)-that specifically mediate protection against PAM than did women receiving a placebo. VSA(PAM)-specific IgG levels depended on the number of IPTp doses received and were sufficiently low to be of clinical concern among multidose recipients. Our data suggest that IPTp should be extended to women of all parities, in line with current World Health Organization recommendations.     

Inhibitory influence of a new steroidal anti-androgen, TZP-4238, on prostatic hyperplasia in the beagle dog.

The effect of a synthetic steroidal anti-androgen, TZP-4238, on spontaneous benign prostatic hyperplasia (BPH) in dogs was investigated. Old male beagle dogs (5-9 years old) were divided into three experimental groups. Group 1 consisted of BPH controls. Groups 2 and 3 received TZP-4238 0.1 mg/kg/day and chlormadinone acetate (CMA) 0.3 mg/kg/day p.o., respectively, for 5 months. In group 1, glandular hyperplasia of the prostate was clearly detected. In contrast, TZP-4238 (Group 2) or CMA (Group 3) produced marked atrophy of the glandular epithelium. In addition, a histopathological study showed that TZP-4238 or CMA medication for 5 months exerted no effect on the testes and the pituitary luteinizing hormone (LH) cells. Therefore, it is suggested that TZP-4238 (0.1 mg/kg) or CMA (0.3 mg/kg) causes regression of spontaneous canine BPH without any histopathological effects on the testes and pituitary LH cells. However, slightly decreased serum testosterone levels were found in TZP-4238-treated animals, due apparently to a direct and/or indirect effect on the testes. Thus, it is suggested that a marginal antigonadotrophic effect cannot be excluded. It is concluded that TZP-4238 is a potent anti-androgen for the treatment of spontaneous canine BPH, without any negative influence on the function of the testes and the pituitary LH cells.     

Determinants of activation by complement of group II phospholipase A2 acting against Escherichia coli.

Prompt killing of many strains of Escherichia coli during phagocytosis in vitro by isolated polymorphonuclear leukocytes (PMN) requires the presence of nonlethal doses of nonimmune serum (B. A. Mannion, J. Weiss, and P. Elsbach, J. Clin. Invest. 86:631-641, 1990). Because this requirement is bypassed in a phospholipase A (PLA)-rich mutant (pldA ) of E. coli, we have examined the effect of serum on bacteria] phospholipid (PL) degradation during phagocytosis of wild-type (pldA+) and PLA-deficient (pldA) E. coli. In parallel with increased killing, nonlethal doses of serum increased the degradation of prelabeled bacterial PL during phagocytosis by two- to fivefold, to nearly the same levels (ca. 50 to 60%) as those produced during phagocytosis of E. coli pldA in the absence of serum. The effects on the E. coli pldA mutant imply that there is a serum-mediated enhancement of granule-associated group II PMN PLA2 activity. At the same doses, serum promoted action against E. coli in the presence of purified rabbit and human group II PLA2 but did not activate bacterial PLA. Related PLA2s that lack specific structural determinants needed for optimal activity against E. coli treated with the bactericidal/permeability-increasing protein (BPI) of PMN are also less active than wild-type group II PLA2 against serum-treated E. coli. Treatment of E. coli with C7- or C9-depleted serum did not enhance bacterial killing or PL degradation during phagocytosis or the action of purified PLA2. In summary, these findings suggest that (i) nonlethal assemblies of the membrane attack complex promote intracellular killing and destruction of E. coli ingested by PMN, in part by promoting the action of granule-associated PLA2 against ingested bacteria, and (ii) structural determinants first implicated in PLA2 action against BPI-treated E. coli are also important in PLA2 action in concert with other host defense systems, such as complement.     

Structural and replicative forms of mitochondrial DNA in tissues from adult and senescent BALB/c mice and Fischer 344 rats

Age-related changes in the structure and replication of mitochondrial DNA (mtDNA) were investigated in different organs from young adult (9–10 months' old) and senescent (28–29 months' old) BALB/c mice and Fischer 344 rats. Total mtDNA from brain, heart, kidney and liver was isolated by centrifugation in ethidium bromide—CsCl gradient and examined for the occurrence of complex forms and replicative intermediates by electron microscopy. The frequency of catenated mtDNA (interlinked molecules containing two or more circular units) varied from about 2.5% to 5% in adult tissues and showed a small increase in the majority of senescent organs. The frequency of double-sized circular molecules, or circular dimers, was very low in adult tissues, with an average of about 0.04% in mice and 0.1% in rats. The frequency of circular dimers increased with aging to 1.9% in mouse brain and 1.5% in rat kidney, with smaller increases (0.4% and 0.7%) in heart mtDNA from both species; there was no significant increase in the other organs. It is suggested that the increase in the frequency of circular dimer mtDNA reflects an overall deterioration of tissue physiology rather than intrinsic senescent changes in the mitochondria. The frequencies and types of the various replicative forms of mtDNA varied significantly according to tissue but not according to species or donor age. The only exception was a significant increase in the frequency of larger replicative forms in senescent mouse liver, to about 20% compared with 12% in adult liver, suggesting an age-related change in the rate of mtDNA replication and/or turnover in this organ.     

Advances of immunohistochemistry and its application to histopathology--studies on liver metastasis of colorectal carcinoma--venous basement membrane laminin

Recently developed immunohistochemical techniques are revolutionizing surgical pathology. Although much has been written describing sensitivity and specificity of reagents in the relationship to given diagnoses, little has been published regarding the evaluation in diagnostic histopathology. Of 4905 cases, immunohistochemical stains were done in our laboratory on 108 cases in 1988. Forty three markers of different kinds were used for 266 specimens. The mean number of markers used in one case was 2.5. Evaluation study of these cases revealed good to limited diagnostic value of immunohistochemical procedures in about 70% of those cases, but no diagnostic value in approximately 30% of the cases. Relationship between venous invasion and basement membrane was investigated to clarify the mechanism of liver metastasis of the colorectal carcinoma by histologically and immunohistochemically using the anti-laminin antibody. Ninety cases of colorectal carcinoma with liver metastasis were studied. Venous invasion was found in 60 of 66 cases of synchronous liver metastasis, and in 14 of 24 cases of metachronous liver metastasis. Venous invasion was considered highly significant to development of liver metastasis, though there was a definite difference in several aspects between the two groups. Well-defined laminin-containing basement membrane was found in the primary tumor in 35 of 90 cases and in metastatic foci in 15 of 28 cases in which examination was performed. It was most frequently seen in well differentiated adenocarcinoma, though statistically not significant compared to moderately differentiated adenocarcinoma. No definite correlation was found between development of liver metastasis and basement membrane associated tumor cells.     

Mutations of the GREAT gene cause cryptorchidism

In humans, failure of testicular descent (cryptorchidism) is one of the most frequent congenital malformations, affecting 1-3% of newborn boys. The clinical consequences of this abnormality are infertility in adulthood and a significantly increased risk of testicular malignancy. Recently, we described a mouse transgene insertional mutation, crsp, causing high intraabdominal cryptorchidism in homozygous males. A candidate gene Great (G-protein-coupled receptor affecting testis descent), was identified within the transgene integration site. Great encodes a seven-transmembrane receptor with a close similarity to the glycoprotein hormone receptors. The Great gene is highly expressed in the gubernaculum, the ligament that controls testicular movement during development, and therefore may be responsible for mediating hormonal signals that affect testicular descent. Here we show that genetic targeting of the Great gene in mice causes infertile bilateral intraabdominal cryptorchidism. The mutant gubernaculae fail to differentiate, indicating that the Great gene controls their development. Mutation screening of the human GREAT gene was performed using DHPLC analysis of the genomic DNA from 60 cryptorchid patients. Nucleotide variations in GREAT cDNA were found in both the patient and the control populations. A unique missense mutation (T222P) in the ectodomain of the GREAT receptor was identified in one of the patients. This mutant receptor fails to respond to ligand stimulation, implicating the GREAT gene in the etiology in some cases of cryptorchidism in humans.     

Immunolocalization of aromatase in human minor salivary glands of the lower lip with primary Sjogren's syndrome

The enzyme aromatase Is Involved In the conversion of androgens to estrogens and in the modulation of various androgenlc and estrogenlc actions. Abnormalities of estrogen metabolism have been postulated to play roles in the development and/or pathophyslology of Sjdgren's syndrome. In the present study, aromatase was immunolocal-ized In 75 cases of Inflammatory disorders of human minor salivary glands of the lower lip. These included cases of primary Sjögren's syndrome (19 cases), of chronic slaladenitis (34 cases) and of mucous extravasation cysts (22 cases), in order to clarify the possible involvement of in situ estrogen production in primary Sjögren's syndrome. Aromatase Immunoreactlvlty was detected In myoepithelial cells of acini and in interstitial cells adjacent to acini and ducts In 13/19 (68%) cases of primary Sjögren's syndrome. In contrast, aromatase expression was detected In only six of 34 (18%) cases of chronic sialadenttis and in seven of 22 (32%) cases of mucous extravasation cyst. These results suggest that Increased aromatase expression in minor salivary glands with primary Sjogren's syndrome in premenopausal women may be involved in the biological features of primary Sjogren's syndrome through the production of estrogens in situ and possibly through the aggravation of the inflammatory reaction.     

Molecular characterisation of glutamate dehydrogenase gene defects in Japanese patients with congenital hyperinsulinism/hyperammonaemia

Congenital hyperinsulinism and hyperammonaemia (CHH) is caused by dysregulation of glutamate dehydrogenase (GDH). We characterised the GDH gene in two Japanese patients with CHH. Patient 1 showed late-onset and mild hypoglycaemic episodes and mild hyperammonaemia, compared with patient 2. In GDH activity of lymphoblasts, patient 1 showed twofold higher basal GDH activity than control subjects and mild insensitivity for GTP inhibition. Patient 2 showed severe insensitivity for GTP inhibition, and similar allosteric stimulation by ADP in the controls. Genetic studies identified heterozygous and de novo L413V and G446D mutations in patients 1 and 2, respectively. COS cell expression study confirmed that both mutations were disease-causing gene. The insensitivity for GTP inhibition in L413V and G446D was emphasised in COS cell expression system as a result of the dosage effect of mutant GDH gene. L413V showed less impairment of GDH than G446D based on biochemical and genetic results, which was consistent with the clinical phenotype. Based on the structure of bovine GDH, G446D was located in GTP binding site of pivot helix and its surroundings, while L413V was located in alpha-helix of antenna-like structure. These different locations of mutations gave different effects on GDH enzyme. The antenna-like structure plays an important role in GDH activity.