Human globin chain separation by isoelectric focusing

Human globin chain separation, a key procedure in the study of hemoglobinopathies, is routinely performed by chromatography on carboxymethylcellulose. This method, though relatively easy and highly reliable, is expensive and time consuming. A new procedure, based on isoelectric focusing, is presented which allows the simultaneous separation of globin chains from multiple samples (at least 20 per gel slab). The method is rapid, inexpensive and can be easily carried out in clinical laboratories, and its high sensitivity allows the identification of radioactive bands even with minute amounts of labelled material. A new phenomenon, called the 'Nonidet P-40 effect', which greatly enhances the separation between gamma and beta chains by binding to these two chains and shifting their pI values in opposite directions, is described.     

Arguments against the prostatic origin of the R-3327 Dunning H tumor

The Dunning tumor, originally described as a carcinoma of the rat dorsal prostate, has for long been used as an experimental model of prostatic cancer. We have recently presented a number of morphological findings that are incompatible with the prostatic origin of the H-subline of the Dunning tumor. In this paper, biochemical and immunohistochemical markers of rat prostate and mammary gland are studied in the R-3327 Dunning H tumor. Pieces of the H tumor were inoculated in male or lactating female rats. The electrophoretic protein pattern of Dunning tumor extracts was more similar to that of the mammary gland than the dorsolateral prostate. Proteins selectively appearing after metabolic labeling in Dunning tumors grown in lactating rats corresponded to labeled proteins in mammary glands from the same animals. Secretory proteins typical of the lateral prostate (SVS II) and dorsal prostate (transglutaminase) could not be detected immunohistochemically in the Dunning tumor. Western blot studies of tumor extracts and slot blot analysis of RNA preparations from the tumor confirmed the absence of SVS II and prostate specific transglutaminase from the Dunning tumor. On the other hand, the presence of mammary gland proteins such as milk fat globule membrane proteins, lactoperoxidase and lactalbumin were detected in the Dunning tumor by immunohistochemistry and Western blotting, but were absent from the dorsolateral prostate. Transferrin-mRNA, expressed in the male urogenital tract and also in the liver and other tissues, was detected in the mammary gland and Dunning tumor, but not in the dorsolateral prostate. The absence of mammary gland secretory beta-casein in the Dunning tumor was related to the elevated Ha-ras oncogene expression in the tumor, previously reported to suppress casein expression. The findings clearly demonstrate that the prostate cannot be the origin of the Dunning tumor, presently being used in prostatic cancer research. The designation prostatic adenocarcinoma for this tumor is therefore invalid. Furthermore, the data support our view that mammary gland might be the origin of the Dunning tumor, although the derivation from the bulbourethral or the parotid glands cannot strictly be excluded.     

Characterization of the oriI and oriII Origins of Replication in Phage-Plasmid P4

In the Escherichia coli phage-plasmid P4, two partially overlapping replicons with bipartite ori sites coexist. The essential components of the oriI replicon are the alpha and cnr genes and the ori1 and crr sites; the oriII replicon is composed of the alpha gene, with the internal ori2 site, and the crr region. The P4 alpha protein has primase and helicase activities and specifically binds type I iterons, present in ori1 and crr. Using a complementation test for plasmid replication, we demonstrated that the two replicons depend on both the primase and helicase activities of the alpha protein. Moreover, neither replicon requires the host DnaA, DnaG, and Rep functions. The bipartite origins of the two replicons share the crr site and differ for ori1 and ori2, respectively. By deletion mapping, we defined the minimal ori1 and ori2 regions sufficient for replication. The ori1 site was limited to a 123-bp region, which contains six type I iterons spaced regularly close to the helical periodicity, and a 35-bp AT-rich region. Deletion of one or more type I iterons inactivated oriI. Moreover, insertion of 6 or 10 bp within the ori1 region also abolished replication ability, suggesting that the relative arrangement of the iterons is relevant. The ori2 site was limited to a 36-bp P4 region that does not contain type I iterons. In vitro, the alpha protein did not bind ori2. Thus, the alpha protein appears to act differently at the two origins of replication.     

Characterization of conjugative transposon Tn5251 of Streptococcus pneumoniae

Abstract Tn5251 belongs to the Tn916-Tn1545 family of conjugative transposons (CT) and was found integrated into CT Tn5252 , to form the composite element Tn5253 of Streptococcus pneumoniae . We show that Tn5251 is identical in structure and size to Tn916 . DNA sequence analysis of a 4,419-bp segment containing the tet(M) gene showed that only 73 nucleotides out of 4,419 were different in the the two CT. Essentially all differences (66 / 73) were clustered in a 688-bp segment of tet(M) , which was 90% identical to Tn916 and 100% identical to the tet(M) genes of Tn1545 from S. pneumoniae and pOZ101 from Neisseria gonorrhoeae . DNA sequence analysis of the Tn5251/Tn5252 junction fragments allowed us (i) to determine Tn5251 termini, (ii) to define the 6-bp coupling sequences flanking the CT, and (iii) to infer the structure of the integration site ( attB ) of Tn5251 into Tn5252 . Conjugal transfer of Tn5251 independent from Tn5253 could not be detected, even if we could show excision and formation of Tn5251 circular intermediates at a level of 5.4 copies per 10⁶ chromosomes.     

Evolutionary pathways to SARS-CoV-2 resistance are opened and closed by epistasis acting on ACE2

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infects a broader range of mammalian species than previously predicted, binding a diversity of angiotensin converting enzyme 2 (ACE2) orthologs despite extensive sequence divergence. Within this sequence degeneracy, we identify a rare sequence combination capable of conferring SARS-CoV-2 resistance. We demonstrate that this sequence was likely unattainable during human evolution due to deleterious effects on ACE2 carboxypeptidase activity, which has vasodilatory and cardioprotective functions in vivo. Across the 25 ACE2 sites implicated in viral binding, we identify 6 amino acid substitutions unique to mouse—one of the only known mammalian species resistant to SARS-CoV-2. Substituting human variants at these positions is sufficient to confer binding of the SARS-CoV-2 S protein to mouse ACE2, facilitating cellular infection. Conversely, substituting mouse variants into either human or dog ACE2 abolishes viral binding, diminishing cellular infection. However, these same substitutions decrease human ACE2 activity by 50% and are predicted as pathogenic, consistent with the extreme rarity of human polymorphisms at these sites. This trade-off can be avoided, however, depending on genetic background; if substituted simultaneously, these same mutations have no deleterious effect on dog ACE2 nor that of the rodent ancestor estimated to exist 70 million years ago. This genetic contingency (epistasis) may have therefore opened the road to resistance for some species, while making humans susceptible to viruses that use these ACE2 surfaces for binding, as does SARS-CoV-2.

This study suggests that ancient events in mammalian cardiovascular evolution determined the host range of SARS-CoV-2 millions of years before the current pandemic. These physiological constraints are so inflexible that escape from SARS-CoV-2 susceptibility would likely have required significant alterations to the human cardiovascular system.     

The microtubule-targeting agent T0070907 induces proteasomal degradation of tubulin

Current microtubule-targeting agents interfere with the regulated assembly of microtubules from α/β tubulin heterodimers but do not markedly alter tubulin levels. Previously, we showed that the compound T0070907 interferes with microtubule function by reversibly decreasing α and β tubulin protein levels by more than 50% in multiple CRC cell lines. Since tubulin levels are generally relatively stable, and cells lack regulatory networks to respond to decreased tubulin levels by increasing synthesis, our result suggested the possibility of cancer therapies that act directly on tubulin homeostasis. The aim of this study was to determine whether T0070907 caused tubulin loss by increasing the degradation rate, and determine the proteases responsible for any increased degradation. T0070907 increased tubulin degradation rates in HT-29 cells. The proteasomal inhibitors MG132, epoxomicin, lactacystin, and ALLN suppressed T0070907-mediated tubulin loss, although epoxomicin and lactacystin were less effective than MG132, even at concentrations that completely inhibited TNFα-induced IκBα degradation. Inhibitors of lysosomal, aggresomal, and calpain-mediated degradation, as well as the caspase inhibitor zVAD-fmk had no effect on tubulin loss, and the cathepsin and calpain inhibitor E64d was unable to increase epoxomicin’s ability to suppress tubulin loss. We conclude that T0070907-induced tubulin degradation proceeds through a proteasome-dependent pathway.     

Basophils infiltrate human gastric mucosa at sites of Helicobacter pylori infection, and exhibit chemotaxis in response to H. pylori-derived peptide Hp(2-20)

Basophils, which are normally confined to the circulation, can migrate to sites of allergic inflammation. Using the specific mAb, BB1, we detected basophil infiltration of the gastric mucosa of Helicobacter pylori-infected patients affected by moderate and severe gastritis. Basophils were not found in H. pylori-free individuals or in subjects with mild gastritis. The H. pylori-derived peptide, Hp(2-20), was a potent basophil chemoattractant in vitro, whereas the control peptide, Hp1, was ineffective. Basophils from peripheral blood of healthy volunteers expressed mRNA for the formyl peptide receptors, N-formyl-peptide receptor (FPR), FPR-like (FPRL)1, and FPRL2. Preincubation of basophils with FMLP or Hp(2-20) caused complete desensitization to a subsequent challenge with homologous stimulus. Incubation of basophils with a low concentration of FMLP, which binds with high affinity to FPR, but not to FPRL1 or FPRL2, did not affect the chemotactic response to Hp(2-20). In contrast, a high concentration of FMLP, which binds to FPRL1 and FPRL2, reduced the chemotactic response to Hp(2-20). The FPR antagonist, cyclosporin H, prevented chemotaxis induced by FMLP, but not by Hp(2-20). Hp(2-20) could be responsible, at least in part, for basophil infiltration of the gastric mucosa of H. pylori-infected patients presumably through the interaction with FPRL1 and FPRL2.     

Research

Gram-positive bacterium Streptococcus gordonii, a human oral commensal, was engineered to display a single-chain Fv (scFv) antibody fragment at the cell surface. The previously developed host-vector system allowed expression of the Guy’s 13 scFv as a fusion with the streptococcal surface protein M6. Surface expression of the 515-amino acid M6/scFv fusion protein was confirmed by Western blot analysis on cellular fractions and flow cytometric analysis. Guy’s 13 scFv was derived from the Guy’s 13 monoclonal antibody, which was raised against streptococcal antigen I/II (SA I/II), the major adhesin of the caries-producing bacterium Streptococcus mutans. Surface plasmon resonance was used to test binding of scFv-expressing S. gordonii to SA I/II. Whole cells of recombinant S. gordonii were found to specifically bind to immobilised SA I/II and binding was inhibited by fluid-phase SA I/II in a dose-dependent manner. Production of a functional scFv in S. gordonii is the first step towards the development of genetically engineered commensal bacteria that, by colonizing mucosal surfaces, may provide the host with sustained delivery of recombinant antibodies.