ISGylation governs the oncogenic function of Ki-Ras in breast cancer.

Aberrant expression of the oncogenic Kirsten-Ras (Ki-Ras) and interferon-stimulated gene 15 (ISG15) pathways is common in breast and other cancers. However, whether these dysregulated pathways cooperate to promote malignancy is not known. This study links Ki-Ras and ISG15 in a previously unidentified regulatory loop that may underlie malignant transformation of mammary cells. We show that oncogenic Ki-Ras regulates the expression of the ISG15 pathway (free ISG15 and ISG15 conjugates), and ISG15, in turn, stabilizes Ki-Ras protein by inhibiting its targeted degradation via lysosomes in breast cancer cells. Disruption of this loop by silencing either Ki-Ras or the ISG15 pathway restored the disrupted cellular architecture, a hallmark feature of most cancer cells. We also demonstrate that ISG15 and UbcH8 (ISG15-specific conjugating enzyme) shRNAs reversed Ki-Ras mutation-associated phenotypes of cancer cells, such as increased cell proliferation, colony formation, anchorage-independent growth in soft agar, cell migration, and epithelial-mesenchymal transition. As UbcH8-silenced breast cancer cells are devoid of ISG15 conjugates but have free ISG15, our results using UbcH8-silenced cells suggest that ISG15 conjugates, and not free ISG15, contributes to oncogenic Ki-Ras transformation. We have thus identified the conjugated form of ISG15 as a critical downstream mediator of oncogenic Ki-Ras, providing a potential mechanistic link between ISG15 and Ki-Ras-mediated breast tumorigenesis. Our findings, which show that inhibition of the ISGylation reverses the malignant phenotypes of breast cancer cells expressing oncogenic Ki-Ras, support the development of ISG15 conjugation inhibitors for treating breast and also other cancers expressing oncogenic Ki-Ras.

Field ecology of toxic Pfiesteria complex species and a conservative analysis of their role in estuarine fish kills.

Within the past decade, toxic Pfiesteria outbreaks have been documented in poorly flushed, eutrophic areas of the largest and second largest estuaries on the U.S. mainland. Here we summarize a decadal field effort in fish kill assessment, encompassing kills related to Pfiesteria (49 major kills in North Carolina estuaries since 1991 and 4 in Maryland estuaries in 1997) and to other factors such as low oxygen stress (79 major fish kills in North Carolina estuaries). The laboratory and field data considered in developing our protocols are described, including toxic Pfiesteria behavior, environmental conditions conducive to toxic Pfiesteria activity, and impacts of toxic clonal Pfiesteria on fish health. We outline the steps of the standardized fish bioassay procedure that has been used since 1991 to diagnose whether actively toxic Pfiesteria was present during estuarine fish kills. Detailed data are given for a 1998 toxic Pfiesteria outbreak in the Neuse Estuary in North Carolina to illustrate of the full suite of diagnostic steps completed. We demonstrate that our conservative approach in implicating toxic Pfiesteria involvement in fish kills has biased in favor of causes other than Pfiesteria. Data are summarized from experiments that have shown stimulation of toxic Pfiesteria strains by nutrient (N, P) enrichment, supporting field observations of highest abundance of toxic strains in eutrophic estuaries. On the basis of a decade of research on toxic Pfiesteria, we present a conceptual model of the seasonal dynamics of toxic strains as affected by changing food resources and weather patterns. We also recommend protocols and research approaches that will strengthen the science of fish kill assessment related to Pfiesteria and/or other causative factors.

Neuroaxonal degeneration in sheep grazing Sorghum pastures.

During the fall of 1992, 250 (10%) of 2,500 Rambouilet cross feeder lambs grazing Sorghum bicolor developed neurologic signs including weakness, ataxia, head shaking, knuckling of the fetlocks, inability to rise, and opisthotonos. One hundred fifteen (46%) of the affected lambs died. Twenty of the surviving lambs exhibited residual neurologic signs of ataxia when stressed. At the same time, 275 (25%) of 1,100 ewes grazing a nearby sudex pasture (S. sudanese x S. bicolor) gave birth to lambs that were weak and unable to rise. Newborn lambs exhibited extensor rigidity and opisthotonos when assisted to a standing position. The dystocias that occurred were due to lambs with contracted limbs (arthrogryposis). All affected lambs died or were euthanized. Histologic examination of the brains of 3 feeder lambs and 9 newborn lambs revealed similar microscopic lesions. The predominant change was the presence of focal axonal enlargements (spheroids) in the proximal segments of axons, which were restricted to the nuclei of the medulla, cerebellum, and midbrain. In addition, the spinal cord contained spheroids in the ventral horn gray matter of the 6 newborns examined. Ultrastructurally, the spheroids were composed of aggregates of neurofilaments, mitochondria, vesicular bodies, and dense bodies bounded by a thin myelin sheath. There was mild gliosis in the more severely affected animals of both groups. There was minimal Wallerian degeneration in the white matter adjacent to affected nuclei in the brain and the ventromedial and dorsolateral funiculi of the spinal cord. This is the first detailed report of Sorghum toxicity in sheep.

Flavin-containing monooxygenase: a major detoxifying enzyme for the pyrrolizidine alkaloid senecionine in guinea pig tissues.

Evidence based on optimal pH, thermal stability, and enzyme inhibition data suggests that the NADPH-dependent microsomal N-oxidation of the pyrrolizidine alkaloid senecionine is carried out largely by flavin-containing monooxygenase in guinea pig liver, lung, and kidney. In contrast, the hepatic microsomal conversion of senecionine to the pyrrole metabolite (+/-)-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP) is catalyzed largely by cytochrome P450. However, the rate of senecionine N-oxide formation (detoxication) far exceeded the rate of DHP formation (activation) in guinea pig liver microsomes over a range of pHs (pH 6.8 to 9.8). In guinea pig lung and kidney microsomes, N-oxide was the major metabolite formed from senecionine with little or no production of DHP. The high rate of detoxication coupled with the low level of activation of senecionine in liver, lung, and kidney may help explain the apparent resistance of the guinea pig to intoxication by senecionine and other pyrrolizidine alkaloids.

Canine distemper virus infection and encephalitis in javelinas (collared peccaries).

Canine distemper virus has been isolated in dog lymphocyte cultures from the brains of three javelinas that became moribund with signs of encephalitis. Canine distemper viral antigen was demonstrated predominantly in neurons and morbillivirus-like structures were seen by electron microscopy in brains of diseased animals. Serological studies suggest that CDV infection may be common in javelinas.

Ocular coccidioidomycosis in a cat.

Enucleation of the right eye was performed on a 12-year-old male Persian cat when therapy for uveitis failed. Histologic examination of the anterior and posterior chambers and the vitreous led to a diagnosis of endophthalmitis caused by Coccidioides immitis infection. The primary focus of infection was not determined. Latex particle agglutination, agar gel immunodiffusion, and complement fixation gave negative results for Coccidioides immitis antibody.

Leptospirosis in rodents from an arid environment.

Rodents (n = 358) were trapped from 6 locations in Arizona, including 2 dairies, 2 swine raising operations, and 2 areas where domestic animal access was limited. Isolates of Leptospira interrogans serovar ballum were obtained from 3 house mice (Mus musculus) trapped in dairies. Leptospira were seen in silver-stained kidney sections of 10.4% of the rodents. The usefulness of serologic data in detecting leptospiral infection in these rodents was uncertain because so few animals yielded isolates that valid comparisons of culture positives to serologic positives were not possible. Titers greater than or equal to 1:160 were obtained to serovars autumnalis, ballum, bratislava, canicola, grippotyphosa, hardjo, icterohaemorrhagiae, and pomona. Nearly 60% of the rodents had microscopic lesions in kidneys, including 20 of 34 (59%) of those in which leptospira were seen.

Repeated sequences and open reading frames in the 3' flanking region of the gene for the RNA subunit of Escherichia coli ribonuclease P.

We have determined the identity of about 700 nucleotides in the 3' flanking region of the gene for M1 RNA, the RNA component of RNase P (EC 3.1.26.5). This region begins with a 113-base-pair segment of DNA, which is repeated approximately 3.5 times. The first repeating unit originates within the gene sequence for the 3' end of mature M1 RNA. The repeating units are highly conserved but diverge considerably after the partial fourth repeat. Segments of sequence homologous to the repeats have been identified both upstream in the M1 RNA transcription unit and downstream from the repeating units. Several overlapping open reading frames, with the potential to encode small basic proteins, have been identified in the repeated sequences. The structure of the M1 RNA gene 3' flanking region is very similar to the corresponding region at the Tyr T locus. In vitro and in vivo, transcription of the M1 RNA gene appears to terminate about 40 nucleotides downstream from the 3' terminus of mature M1 RNA. Therefore, an RNA processing event is involved in the biosynthesis of M1 RNA.

Nucleotide sequence of the gene encoding the RNA subunit (M1 RNA) of ribonuclease P from Escherichia coli.

The gene encoding the RNA subunit (M1 RNA) of RNAase P (EC 3.1.26.5) from Escherichia coli has been isolated, and its complete nucleotide sequence, including flanking regions, has been determined. The promoter region, similar to others near genes under stringent control, and the site of transcription termination have been identified. The transcript from the gene (M1 RNA) can be drawn in a secondary structure that has approximately 60% G-C base pairs. One hairpin loop of this hypothetical structure has five contiguous nucleotides complementary to invariant nucleotides in the TpsiCG loop of all E. coli tRNAs. The M1 gene, when subcloned in the plasmid pBR325, can be amplified. It directs production of functional M1 RNA. In an E. coli strain thermosensitive for RNAase P function, the size of the gene transcript is the same as in wild-type E. coli, but less mature M1 RNA is made in the mutant cells.

Polycythemia vera and agnogenic myeloid metaplasia.

Age complications bring these patients to primary physicians more frequently than would be expected from incidence or prevalence statistics alone. Therapy is symptomatic. Termination in acute leukemia occurs in both conditions and the risk may be increased by radiation or myelosuppressive drugs. Careful management can prolong survival in polycythemia vera and improve the quality of life in both conditions.

Lipogenesis and the synthesis and secretion of very low density lipoprotein by avian liver cells in nonproliferating monolayer culture. Hormonal effects.

The nonproliferating chicken liver cell culture system described yields cell monolayers with morphological and lipogenic properties characteristic of the physiological-nutritional state of donor animals. Synthesis and secretion of fatty acid, cholesterol, and very low density lipoprotein (VLDL) occur at in vivo rates and respond to hormones and agents which affect these processes in vivo. Cells derived from fed chickens maintain high rates of synthesis of fatty acid and cholesterol for several days if insulin is present in the medium. High rates of fatty acid synthesis are correlated with the appearance of membrane-enclosed triglyceride-rich vesicles in the cytoplasm; deletion of insulin causes a decrease (T1/2 = 22 h) in fatty acid synthetic activity. Addition of glucagon or cyclic AMP (cAMP) causes an immediate cessation of fatty acid synthesis and blocks the appearance of the triglyceride-rich vesicles. Fatty acid synthesis in liver cells prepared from fasted chickens is less than 5% that of cells from fed animals. After 2-3 days in culture with serum-free medium containing insulin +/- triiodothyronine, fatty acid synthesis is restored to normal; glucagon or dibutyryl cAMP blocks this recovery. Liver cells derived from estradiol-treated chickens synthesize and secrete VLDL for at least 48 h in culture. Electron micrographs of these cells reveal more extensive development of the rough endoplasmic reticulum and Golgi complex compared to cells from untreated chickens. Whereas [3H]leucine incorporation into total protein is unaffected by estrogen treatment, [3H]leucine incorporation into cellular and secreted immunoprecipitable VLDL is markedly increased indicating specific activation of VLDL apopeptide synthesis; 8-10% of the labeled protein synthesized and secreted is VLDL. Dodecyl sulfate-acrylamide gel electrophoresis of immunoprecipitated 3H-VLDL reveals three major apopepetides of 300,000, 11,000, and 8,000 daltons corresponding to those of purified chicken VLDL.

Ankylosing spondylitis with selective IgA deficiency and a circulating anticoagulant.

A patient with ankylosing spondylitis was found to have selective IgA deficiency and a non-heparin, immediate-acting antithrombin (antithrombin V). T cells were decreased, and serum IgG was increased. In vitro synthesis of IgG by peripheral blood lymphocytes was very high. This association of ankylosing spondylitis with the T cell and protein abnormalities is probably fortuitous but does demonstrate that severe spondylitis may evolve in the absence of IgA.

Coccidioidomycosis in a California sea lion (Zalophus californianus).

Coccidioidomycosis in an adult male California sea lion (Zalophus californianus) is described. The animal was housed in a zoo in Tucson, Arizona, for approximately 5 years. This is believed to be the first reported case of coccidioidomycosis in a marine mammal.

Partial purification and characterization of aspartate aminotransferases from seedling oat leaves.

As relatively little information is available on the properties of aspartate aminotransferase from photosynthetic tissue, isolation and characterization of the two major electrophoretically distinct forms of this enzyme from seedling oat leaf homogenates were undertaken. These two forms are designated I for the more anionic form and II for the less anionic form. Form I, 80 to 90% of the total activity, has been purified to a specific activity of 120 mumol/min/mg of protein (1100-fold) and is estimated to be 90 to 95% homogeneous, as judged by analytical polyacrylamide gel electrophoresis. Form II, 10 to 20% of the total activity, has been purified to a specific activity of approximately 6 mumol/min/mg of protein (300-fold). Both forms exhibit optimal activity at pH 7.5. Michaelis constants do not differ greatly between forms I and II and are similar to those reported for the pig heart cytosolic enzyme as well as aspartate aminotransferase from other plant sources. A molecular weight of 130,000 for the purified aspartate aminotransferase I was estimated by sedimentation equilibrium centrifugation; molecular weights of the two forms are similar as estimated by sucrose density gradient centrifugation. No activation by pyridoxal phosphate has been observed during purification.