Characterization of maternal small lymphocyte subsets during allogeneic pregnancy in the mouse

Possible changes in the maternal small lymphocyte subsets within the bone marrow, spleen, paraaortic lymph nodes (PALN, draining the uterus) and blood were studied during allogeneic (CBA♀ × C57BL♂) vs syngeneic (CBA♀ × CBA♂) pregnancy from a radioautographic examination of surface markers: IgM on B cells, Thy-1 antigen on T cells and an absence of either marker on null cells. The temporal patterns of changes in the absolute numbers of these small lymphocyte subsets in different lymphoid organs were qualitatively similar for both types of pregnancies, but these changes were more marked in the allogeneic type. Null cell numbers increased initially in the marrow (Days 6–7), then in the blood (Days 8–9) and finally in the spleen and PALN (Day 14). The T cell content initially declined and subsequently recovered in all lymphoid organs. B cell numbers remained essentially unchanged. While the changes common to both kinds of pregnancy may reflect a maternal response to fetal antigens, the more pronounced alterations seen during allogeneic pregnancy may result from an additional response to paternal type alloantigens including H-2. These findings are similar to those reported by us for tumor-transplanted mice.     

Immunoprecipitation of Native Botulinum Neurotoxin Complexes from Clostridium botulinum Subtype A Strains

Botulinum neurotoxins (BoNTs) naturally exist as components of protein complexes containing nontoxic proteins. The nontoxic proteins impart stability of BoNTs in the gastrointestinal tract and during purification and handling. The two primary neurotoxin complexes (TCs) are (i) TC1, consisting of BoNT, nontoxin-nonhemagglutinin (NTNH), and hemagglutinins (HAs), and (ii) TC2, consisting of BoNT and NTNH (and possibly OrfX proteins). In this study, BoNT/A subtypes A1, A2, A3, and A5 were examined for the compositions of their TCs in culture extracts using immunoprecipitation (IP). IP analyses showed that BoNT/A1 and BoNT/A5 form TC1s, while BoNT/A2 and BoNT/A3 form TC2s. A Clostridium botulinum host strain expressing recombinant BoNT/A4 (normally present as a TC2) from an extrachromosomal plasmid formed a TC1 with complexing proteins from the host strain, indicating that the HAs and NTNH encoded on the chromosome associated with the plasmid-encoded BoNT/A4. Strain NCTC 2916 (A1/silent B1), which carries both an ha silent bont/b cluster and an orfX bont/a1 cluster, was also examined. IP analysis revealed that NCTC 2916 formed only a TC2 containing BoNT/A1 and its associated NTNH. No association between BoNT/A1 and the nontoxic proteins from the silent bont/b cluster was detected, although the HAs were expressed as determined by Western blotting analysis. Additionally, NTNH and HAs from the silent bont/b cluster did not form a complex in NCTC 2916. The stabilities of the two types of TC differed at various pHs and with addition of KCl and NaCl. TC1 complexes were more stable than TC2 complexes. Mouse serum stabilized TC2, while TC1 was unaffected.     

Fluorescence studies on erythrocyte membrane isolated fromPlasmodium berghei infected mice

The erythrocyte host cell plays a key role in the well defined developmental stages of the malarial parasite growth and propogation in the erythrocyte cycle of malaria. The host cell serves the parasites by supplying metabolites and removing the catabolites produced by the obligatory parasites. It has been observed that the plasma membrane of the infected cells show a substantially higher fluidity probably due to the depletion of cholesterol content from the host cell. The protein component of the membrane is also modulated due to the insertion of new polypeptides of the parasitic origin, which confers upon it new antigenic properties. We have studied the membrane fraction isolated from mice erythrocytes infected withPlasmodium berghei using fluorescent probes like DPH, ANS and series of fluorenyl fatty acids, which permit depth dependent analysis of membrane. We have observed that there is a marked difference in the fluorescence emission wavelength maximum, the dissociation constant K_d of ANS when bound to normal and infected erythrocytes, though relatively small differences are observed in the fluorescence polarisation values of the two cell types. The fluorenyl fatty acids also show the differences when bound to normal and infected erythrocytes, indicating that either they are in a different environment or they have differing binding properties to the two cell types.Abbreviations DPH 1,6-Diphenyl-1,3,5-Hexatriene - ANS 8-Anilino-napthalene Sulfonic Acid - C2A-FL 2-Fluorenyl-acetic Acid - C4A-FL 2-Fluorenyl-butyric Acid - C6A-FL 2-Fluorenyl-hexanoic Acid - C8A-FL 2-Fluorenyl-octanoic Acid     

Cis-trans isomerization of proline in the peptide (his 105-Val 124) of ribonuclease a containing the primary nucleation site

Conformational energy calculations have been carried out to determine the relative stabilities of the C-terminal sequence 105–124 of ribonuclease A, withcis andtrans forms, respectively, of Asn 113-Pro 114. Thecis form of Pro 114 is the one that occurs in the native protein. This peptide contains the sequence 106–118, which, on the basis of both theoretical and experimental studies, is thought to constitute the primary nucleation site for the folding of ribonuclease A. It is shown that both conformations of the isolated peptide (with Pro 114 in thecis andtrans forms, respectively) are of approximately equal stability. Both forms have similar conformations from residues 105–110 and 118–124, while they differ in the bend region involving residues 111–117. Calculations have also been carried out to deduce the possible low-energy paths for the interconversion between thecis andtrans forms of both Pro 114 and Pro 117. It is shown that there are two low-energy paths (with a minimum activation energy of 16.5 kcal/mole) for the interconversion of Pro 114. Attractive nonbonded interaction energies stabilize the transition state on these paths. Only one relatively low-energy path (with an activation energy of 18 kcal/mole) could be found for the isomerization of Pro 117, which occur in thetrans form in the native protein; in this case, allcis forms have significantly higher energy than thetrans form. These calculations thus show that native-like forms for the isolated peptide can exist with Pro 114 in either thecis or thetrans form and that these forms are readily interconvertible.     

Are there biases in biopsy sampling? Potential drivers of sex ratio in projectile biopsy samples from two small delphinids

Molecular assays were used to determine the sex of 1,294 biopsied common dolphins (658 long‐beaked common dolphins, Delphinus capensis, and 636 short‐beaked common dolphins, D. delphis) in the Southern California Bight. Sex ratio differed substantially between the two species; females comprised 241 (36.6%) of D. capensis samples and 410 (64.5%) of D. delphis samples. All biopsies were taken either from a large research ship or from a small, rigid‐hull inflatable boat (RHIB) launched from the larger ship. When conducting replicate biopsy effort on the same schools from each vessel/platform (“Tandem Biopsy Sampling”), we found evidence that disproportionately more female D. capensis were biopsied from the RHIB than from the ship but the same was not true for D. delphis. We suspect that these results are driven by bowriding‐behavior differences between the two species. Biopsy duration, geographic location, school size, and Julian date were considered as potential covariates with sex ratio; geographic location was the only one to show strong evidence of correlation. This study also presents an alternative to the erroneous practice of comparing sex ratios to a theoretical assumption of parity (i.e., 50:50 sex ratio) when researchers avoid sampling animals paired with calves.     

Detection of cancer-associated miRNA using a fluorescence switch of AgNC@NA and guanine-rich overhang sequences

DNA-templated silver nanoclusters (AgNC@DNA) are a novel type of nanomaterial with advantageous optical properties. Only a few atoms in size, the fluorescence of nanoclusters can be tuned using DNA overhangs. In this study, we explored the properties of AgNCs manufactured on a short single-stranded (dC)₁₂ when adjacent G-rich sequences (dG_N, with N = 3–15) were added. The ‘red’ emission of AgNC@dC₁₂ with λ_MAX = 660 nm dramatically changed upon the addition of a G-rich overhang with N_G = 15. The pattern of the emission–excitation matrix (EEM) suggested the emergence of two new emissive states at λ_MAX = 575 nm and λ_MAX = 710 nm. The appearance of these peaks provides an effective way to design biosensors capable of detecting specific nucleic acid sequences with low fluorescence backgrounds. We used this property to construct an NA-based switch that brings AgNC and the G overhang near one another, turning ‘ON’ the new fluorescence peaks only when a specific miRNA sequence is present. Next, we tested this detection switch on miR-371, which is overexpressed in prostate cancer. The results presented provide evidence that this novel fluorescent switch is both sensitive and specific with a limit of detection close to 22 picomoles of the target miR-371 molecule.     

Structural basis of sequestration of the anti-Shine-Dalgarno sequence in the Bacteroidetes ribosome

Genomic studies have indicated that certain bacterial lineages such as the Bacteroidetes lack Shine-Dalgarno (SD) sequences, and yet with few exceptions ribosomes of these organisms carry the canonical anti-SD (ASD) sequence. Here, we show that ribosomes purified from Flavobacterium johnsoniae, a representative of the Bacteroidetes, fail to recognize the SD sequence of mRNA in vitro. A cryo-electron microscopy structure of the complete 70S ribosome from F. johnsoniae at 2.8 Å resolution reveals that the ASD is sequestered by ribosomal proteins bS21, bS18 and bS6, explaining the basis of ASD inhibition. The structure also uncovers a novel ribosomal protein—bL38. Remarkably, in F. johnsoniae and many other Flavobacteriia, the gene encoding bS21 contains a strong SD, unlike virtually all other genes. A subset of Flavobacteriia have an alternative ASD, and in these organisms the fully complementary sequence lies upstream of the bS21 gene, indicative of natural covariation. In other Bacteroidetes classes, strong SDs are frequently found upstream of the genes for bS21 and/or bS18. We propose that these SDs are used as regulatory elements, enabling bS21 and bS18 to translationally control their own production.