Detection of Apparent Early Rabies Infection by LN34 Pan-Lyssavirus Real-Time RT-PCR Assay in Pennsylvania.

The Pennsylvania Department of Health Bureau of Laboratories (PABOL) tested 6855 animal samples for rabies using both the direct fluorescent antibody test (DFA) and LN34 pan-lyssavirus reverse transcriptase quantitative PCR (RT-qPCR) during 2017-2019. Only two samples (0.03%) were initially DFA negative but positive by LN34 RT-qPCR. Both cases were confirmed positive upon re-testing at PABOL and confirmatory testing at the Centers for Disease Control and Prevention by LN34 RT-qPCR and DFA. Rabies virus sequences from one sample were distinct from all positive samples processed at PABOL within two weeks, ruling out cross-contamination. Levels of rabies virus antigen and RNA were low in all brain structures tested, but were higher in brain stem and rostral spinal cord than in cerebellum, hippocampus or cortex. Taken together, the low level of rabies virus combined with higher abundance in more caudal brain structures suggest early infection. These cases highlight the increased sensitivity and ease of interpretation of LN34 RT-qPCR for low positive cases.

Chronic morphine acts via a protein kinase Cgamma-G(beta)-adenylyl cyclase complex to augment phosphorylation of G(beta) and G(betagamma) stimulatory adenylyl cyclase signaling.

Chronic morphine augments protein kinase C (PKC) phosphorylation of G(beta), which enhances the potency of G(betagamma) to stimulate adenylyl cyclase II (ACII) activity. The present study demonstrates an in vivo association between phosphorylated G(beta) and a specific PKC isoform, PKCgamma. We investigated the association of G(beta) and PKCgamma by assessing the ability of anti-PKCgamma antibodies to co-immunoprecipitate G(beta) from (32)P-radiolabeled Chinese Hamster Ovary cells stably transfected with a mu-opioid receptor (MOR-CHO). PKCgamma immunoprecipitate (IP) obtained from MOR-CHO membranes contained radiolabeled signals of approximately equals 33 and 36--38 kDa that were subsequently identified as G(beta)(s). Chronic morphine significantly increased ( approximately equals 75%) the magnitude of (32)P incorporated into G(beta) present in PKCgamma IP. This suggests that G(beta) is an in vivo substrate for PKCgamma, which mediates the chronic morphine-induced increment in G(beta) phosphorylation. In order to evaluate AC as a putative effector for phosphorylated G(betagamma), its presence in IP obtained using anti-AC antibodies was evaluated. Autoradiographic analyses of AC IP also revealed the presence of phosphorylated G(beta)(s), the magnitude of which was significantly enhanced ( approximately equals 60%) following chronic morphine treatment. This indicates that phosphorylated G(betagamma) associates and presumably interacts in vivo with AC, indicating that it is a target for the enhanced phosphorylated G(betagamma) that is generated following chronic morphine treatment. This would contribute to the previously observed shift from predominantly G(ialpha) inhibitory to G(betagamma) stimulatory AC signaling following chronic morphine. The PKCgamma-G(beta)-AC complex identified in this study provides an organizational framework for understanding the well-documented participation of PKCgamma in opioid tolerance-producing mechanisms.

Biochemical demonstration of mu-opioid receptor association with Gsalpha: enhancement following morphine exposure.

Biochemical data indicate mu-opioid receptor (MOR) coupling predominantly to the G(i) and G(o) family. Additionally, MOR coupling to G(s) is suggested by pharmacological assessments that have revealed excitatory MOR effects, which are resistant to pertussis toxin and sensitive to cholera toxin. However, biochemical evidence for such interactions remains elusive; G(salpha) has not been shown to be present in immunoprecipitate obtained using anti-MOR antibodies. In the current study, the presence of MOR in immunoprecipitate obtained with anti-G(salpha ) antibodies was investigated using Chinese hamster ovary cells stably transfected with MOR (MOR-CHO). MOR Western analyses of opioid naive MOR-CHO membranes immunoprecipitated using anti-G(salpha) antibodies reveal the presence of an approximately 75-80 kDa MOR species. Interestingly, acute and chronic morphine treatment markedly enhances the magnitude of MOR that co-immunoprecipitates with G(salpha), despite the concomitant down-regulation of membrane MOR protein. Enhanced co-precipitation of MOR with G(salpha) occurs without a concomitant increase in the immunoprecipitated G(salpha) protein indicating their increased association. In contrast, chronic morphine diminishes the co-immunoprecipitation of MOR with G(ialpha). Moreover, although only a single MOR species co-immunoprecipitated with G(salpha), MOR Western analysis of MOR-CHO membranes as well as immunoprecipitate obtained with either anti-MOR or anti-G(ialpha) antibodies reveals the presence of multiple molecular mass species of MOR. These data reveal the existence of a subset of MORs whose association with G(salpha) can be enhanced by morphine exposure. Notably, the regulation by chronic morphine of MOR association with G(salpha) and G(ialpha) is reciprocal. The relevance of MOR-Gs(alpha) coupling to opioid tolerance formation is discussed.

The half-life of the transcript encoding the folate receptor alpha in KB cells is reduced by cytosolic proteins expressed in folate-replete and not in folate-depleted cells.

The KB cell, a transformed human cell line, constitutively expresses a high level of the glycosylphosphatidylinositol (GPI) anchored folate receptor protein alpha (FR alpha) and thereby can grow in medium containing <1 nM folate. When transferred from a folate-replete (FR) medium to one folate-deficient (FD), intracellular folate diminishes about 50-fold and expression of the FR alpha increases 6-fold. This up-regulation is mediated by a 4.5-fold increase in the initial transcription rate and a 2.4-fold prolongation of the mRNA half-life that together provide a higher level of the steady-state mRNA abundance. An RNA gel -shift assay of a 5' region of the mRNA that includes all of the non-coding and 24 nt of coding sequence, and a 3' region comprised only of coding sequence, identified unique complexes with cytosolic proteins from the FR-KB cells that were not observed with the cytosol from FD-KB cells. It appears, therefore, that expression of these folate-dependent cytosolic trans-active proteins function to maintain a shorter half-life of the mRNA in the FR-KB cells by binding to 5' and 3' cis elements, reducing the stability of this transcript.

Congenital transcobalamin II deficiency due to errors in RNA editing.

Transcobalamin II (TCII) is a plasma protein essential for the transport and cellular uptake of vitamin B12 (B12; cobalamin, Cbl). Congenital deficiency of functional TCII is an autosomal recessive genetic disorder that results in clinical B12 deficiency usually within several months following birth. In this report, we describe the molecular basis for TCII deficiency in two patients who developed a megaloblastic anemia in early infancy. The serum of both patients contained immunoreactive TCII that did not bind [57Co]Cbl. The fibroblasts from each patient secreted a similarly nonfunctional TCII, yet full-length TCII transcripts were identified by Northern blot. Overlapping cDNA fragments were generated by reverse transcription-polymerase chain reaction and several mutations were identified in the coding region of the cDNA, one of which was common to both patients. However, amplification of the corresponding regions of the gene from genomic DNA failed to identify these mutations. These findings were confirmed by replicate analyses and support the proposal that a variance in RNA editing is the likely mechanism for the mutations that resulted in the expression of a nonfunctional TCII protein in these patients.