The B1-immunoreactive proteins (B1-IPs) are major secretory products of rat submandibular gland acinar-cell progenitors, and are also produced by neonatal and adult rat sublingual and parotid glands. In order to characterize the B1-IPs, we have previously isolated cDNA clones encoding rat parotid secretory protein (PSP; the predominant parotid B1-IP) and the related clone ZZ3, which is developmentally regulated in the neonatal submandibular gland. The remainder of the B1-IPs were uncharacterized. This report demonstrates that all of the B1-IPs are derived from the PSP and ZZ3 transcripts. Molecular cloning and Western-blot analyses using PSP- and ZZ3-specific antisera show that, of the B1-IPs, only PSP and neonatal submandibular gland protein A (SMGA) are products of the Psp gene. This finding corrects our previous assertion that SMGA is derived from ZZ3. Neonatal submandibular gland proteins B1 and B2, as well as apparent Mr 26000-28000 and Mr 18000-20000 forms in submandibular, sublingual and parotid glands, are derived from the gene encoding ZZ3 by differential N-glycosylation and by proteolytic cleavage. The apparent Mr 18000-20000 proteolytic products are significant in secretion product collected in vitro, but rare in gland homogenate and submandibular/sublingual saliva. The gene encoding ZZ3 has been named Smgb. Psp and Smgb are regulated similarly in the developing submandibular gland, but differently in the sublingual and parotid glands. The expression pattern of Psp is conserved between rat and mouse. However, no evidence for proteins derived from an Smgb-like gene was observed in neonatal mouse submandibular or sublingual glands.

CK2α is a ubiquitous, well-studied kinase that is a target for small-molecule inhibition, for treatment of cancers. While many different classes of adenosine 5′-triphosphate (ATP)-competitive inhibitors have been described for CK2α, they tend to suffer from significant off-target activity and new approaches are needed. A series of inhibitors of CK2α has recently been described as allosteric, acting at a previously unidentified binding site. Given the similarity of these inhibitors to known ATP-competitive inhibitors, we have investigated them further. In our thorough structural and biophysical analyses, we have found no evidence that these inhibitors bind to the proposed allosteric site. Rather, we report crystal structures, competitive isothermal titration calorimetry (ITC) and NMR, hydrogen–deuterium exchange (HDX) mass spectrometry, and chemoinformatic analyses that all point to these compounds binding in the ATP pocket. Comparisons of our results and experimental approach with the data presented in the original report suggest that the primary reason for the disparity is nonspecific inhibition by aggregation.

Clostridium sporogenes, Peptostreptococcus anaerobius, and Bacteroides asaccharolyticus have been reported to react in the Culturette Brand Rapid Latex Test (Marion Scientific, Div. Marion Laboratories, Inc., Kansas City, Mo.) for Clostridium difficile. From the results of this study we showed that C. sporogenes and P. anaerobius produce a protein which is very similar biochemically and immunologically to the protein of C. difficile that is detected by the test. Thus, the positive latex reactions observed with C. sporogenes and P. anaerobius are due to a cross-reactive protein. We did not detect this cross-reactive protein in filtrates from B. asaccharolyticus, indicating that this bacterium reacts with the latex reagent by some other mechanism. We cloned the C. difficile gene that codes for the cross-reactive protein and showed that the protein produced by the recombinant organism is nontoxic and distinct from toxin A, thus confirming our earlier findings. Recently, a commercial latex test (the Culturette Brand Rapid Latex Test; Marion Scientific, Div. Marion Laboratories, Inc., Kansas City, Mo.) became available for the clinical detection of Clostridium difficile, the etiologic agent of pseudomembranous colitis. Initially, the test was marketed for the detection of toxin A. Results from our laboratory and other laboratories have shown, however, that the test detects an antigen that is distinct from the toxins (1, 5, 11, 14). The latex-reactive antigen is produced by all of the toxigenic and nontoxigenic strains of C. difficile which we have examined (D. Lyerly and T. Wilkins, unpublished data). Further evaluation of the latex test has revealed that Clostridium sporogenes and proteolytic Clostridium botulinum, which are indistinguishable with the exception of neurotoxin production (17), react in the test (5; B.

Virus-like particles are an emerging class of nano-biotechnology with the Tobacco Mosaic Virus (TMV) having found a wide range of applications in imaging, drug delivery, and vaccine development. TMV is typically produced in planta, and, as an RNA virus, is highly susceptible to natural mutation that may impact its properties. Over the course of 2 years, from 2018 until 2020, our laboratory followed a spontaneous point mutation in the TMV coat protein—first observed as a 30 Da difference in electrospray ionization mass spectrometry (ESI–MS). The mutation would have been difficult to notice by electrophoretic mobility in agarose or SDS-PAGE and does not alter viral morphology as assessed by transmission electron microscopy. The mutation responsible for the 30 Da difference between the wild-type (wTMV) and mutant (mTMV) coat proteins was identified by a bottom-up proteomic approach as a change from glycine to serine at position 155 based on collision-induced dissociation data. Since residue 155 is located on the outer surface of the TMV rod, it is feasible that the mutation alters TMV surface chemistry. However, enzyme-linked immunosorbent assays found no difference in binding between mTMV and wTMV. Functionalization of a nearby residue, tyrosine 139, with diazonium salt, also appears unaffected. Overall, this study highlights the necessity of standard workflows to quality-control viral stocks. We suggest that ESI–MS is a straightforward and low-cost way to identify emerging mutants in coat proteins.