Using a HeLa cell line stably transfected with the tat gene from human immunodeficiency virus type 1, we have found that the expression of the regulatory Tat protein suppresses the expression ofcellular Mn-containing superoxide dismutase (Mn-SOD). This enzyme is one of the cell's primary defenses against oxygen-derived free radicals and is vital for maintaining a healthy balance between oxidants and antioxidants. The parental HeLa cells expressed nearly equivalent amounts of Cu,Zn-and Mn-SOD isozymes. Those cels expressing the Tat protein, however, contained 52% less Mn-SOD activity than parental cells, whereas that of the Cu,Zn enzyme was essentially unchanged. The steady-state levels of Mn-SOD-specific RNAs were also lower in the HeLa-tatcell line than in the parental line. No difference was seen in the steady-state levels of Cu,Zn-SOD-specific RNAs. In addition to the decreased Mn-SOD activity, HeLa-tatceDls showed evidence of increased oxidative stress. Carbonyl proteins were markedly higher, and total cellular sulfhydryl content decreased in ceDl extracts at a faster rate, probably reflecting ongoing lipid peroxidation. HeLa and HeLa-tat extracts were incubated with radiolabeled Mn-SOD transcripts, and the reaction products were subjected to UV crosslinking, digestion with ribonuclease A, and electrophoretic analysis. The results suggest a direct interaction between Tat protein and Mn-SOD gene transcripts.

In situ hybridization (ISH) and immunohistochemistry (IHC) are essential tools to characterize SARS-CoV-2 infection and tropism in naturally and experimentally infected animals and also for diagnostic purposes. Here, we describe three RNAscope ®-based ISH assays targeting the ORF1ab, spike, and nucleocapsid genes and IHC assays targeting the spike and nucleocapsid proteins of SARS-CoV-2. Handling editor: John Ziebuhr.

Toxoplasma gondii is a zoonotic protozoan pathogen that infects many endothermic vertebrates, including humans; the domestic cat and other felids serve as the definitive host. Macropodids are considered highly susceptible to toxoplasmosis. Here, we describe the clinical, pathologic, and immunohistochemical findings of an outbreak of systemic toxoplasmosis in a mob of 11 red kangaroos ( Macropus rufus), with high morbidity (73%) and mortality (100%) rates. Affected animals had either severe and rapidly deteriorating clinical conditions or sudden death, which was correlated with widespread necrotizing lesions in multiple organs and intralesional T. gondii organisms identified via MIC3-specific immunohistochemistry and confirmed by REP529-specific rtPCR. Quantification of parasites demonstrated the highest parasite density in pulmonary parenchyma compared with other tissues. Our study highlights the continued importance of this severe condition in Australian marsupials.

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Peptides from rat liver aldehyde dehydrogenase (AIDH) induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) treatment match the AIDH structure from HTC rat hepatoma cells (HTC-AIDH) at all positions examined, indicating induction of the same gene product by two independent routes. This 452 amino acid residue, class 3 AIDH structure differs substantially from the 500-residue AIDH structures isolated from normal liver cytosol (class 1) and mitochondria (class 2). Despite a 29.8% identity in 429 overlapping amino acids vs the human class 1 enzyme (27.7% vs class 2), neither the N- nor C-termini coincide, and gaps are introduced to optimize the alignment. Two residues placed in the active site of human liver AIDH by chemical modification, Cys-302 and Glu-268, are conserved in class 3 AIDH as Cys-243 and Glu-209. Cys-243/302 is the only cysteine residue conserved in all known AIDH structures. Gly-245 and Gly-250 of class 1/2 AIDHs, fitting the patterns of glycine residues in coenzyme binding fold of other dehydrogenases, are also conserved. Otherwise, Cys-49, Cys-162, and Glu-487, to which functional importance has also been ascribed, are not retained in the class 3 structure. Overall, a high conservation of Gly, Pro, and Trp and similar patterns of predicted secondary structure indicate general conservation of tertiary structure, as noted with other distantly related proteins. Three exon boundaries from the human liver mitochondria AIDH gene directly correspond to the N-terminus of the rat class 3 protein and to two of the gaps in the alignment.(ABSTRACT TRUNCATED AT 250 WORDS)