Asymmetric cell division in Mycobacterium tuberculosis and its unique features

Recently, several reports showed that about 80 % of mid-log phase Mycobacterium smegmatis, Mycobacterium marinum, and Mycobacterium bovis BCG cells divide symmetrically with 5–10 % deviation in the septum position from the median. However, the mode of cell division of the pathogenic mycobacterial species, Mycobacterium tuberculosis, remained unclear. Therefore, in the present study, using electron microscopy, fluorescence microscopy of septum- and nucleoid-stained live and fixed cells, and live cell time-lapse imaging, we show the occurrence of asymmetric cell division with unusually deviated septum/constriction in 20 % of the 15 % septating M. tuberculosis cells in the mid-log phase population. The remaining 80 % of the 15 % septating cells divided symmetrically but with 2–5 % deviation in the septum/constriction position, as reported for M. smegmatis, M. marinum, and M. bovis BCG cells. Both the long and the short portions of the asymmetrically dividing M. tuberculosis cells with unusually deviated septum contained nucleoids, thereby generating viable short and long cells from each asymmetric division. M. tuberculosis short cells were acid fast positive and, like the long cells, further readily underwent growth and division to generate micro-colony, thereby showing that they were neither mini cells, spores nor dormant forms of mycobacteria. The freshly diagnosed pulmonary tuberculosis patients’ sputum samples, which are known for the prevalence of oxidative stress conditions, also contained short cells at the same proportion as that in the mid-log phase population. The probable physiological significance of the generation of the short cells through unusually deviated asymmetric cell division is discussed.     

Omasal ciliated protozoa in cattle, bison, and sheep

Omasal contents were collected from slaughtered cattle (n = 54), bison (n = 15), and sheep (n = 40) to determine numbers and generic distribution of ciliated protozoa. Total protozoan numbers were significantly lower in omasal contents than in ruminal contents of all three species, but the percent composition of all protozoan genera was similar between omasal and ruminal populations. The highest numbers of omasal protozoa found were 7.61 X 10(5)/g in cattle, 7.01 X 10(5)/g in bison, and 1.29 X 10(6)/g in sheep. Omasal dry matter was significantly higher than ruminal dry matter in all species and ranged up to 51.5% in cattle fed high-concentrate diets. The omasal pH was similar to the ruminal pH in all species. The number of omasal laminae averaged 149, 145, and 74 for cattle, bison, and sheep, respectively. Although protozoan concentrations in omasal contents were approximately 80% lower than those in ruminal contents, the omasum harbored relatively high numbers of ciliated protozoa. The resident omasal protozoa are extremely difficult to remove, particularly in cattle, and apparently are responsible for reinoculating transiently defaunated rumens.     

Two type I restriction enzymes from Salmonella species. Purification and DNA recognition sequences

We have purified the type I restriction enzymes SB and SP from Salmonella typhimurium and S. potsdam, respectively, and determined the DNA sequences that they recognize. These sequences resemble those previously determined for the type I enzymes, EcoB, EcoK and EcoA, in that the specific part of the sequence is divided into two domains by a spacer of non-specific sequence that has a fixed length for each enzyme. Two main differences from the previously determined sequences are seen. Both of the new sequences are degenerate and one of them, SB, has one trinucleotide and one pentanucleotide-specific domain rather than the trinucleotide and tetranucleotide domains seen for all of the other enzymes. The only conserved features of the recognition sequences are the adenosyl residues that are methylated in the modification reaction. For all of the enzymes these are situated ten or 11 base-pairs apart, one on each strand of the DNA. This suggests that the enzymes bind to DNA along one face of the double helix making protein-DNA interaction in two successive major grooves with most of the non-specific spacer sequence in the intervening minor groove.     

Lysine acetylation of the Mycobacterium tuberculosis HU protein modulates its DNA binding and genome organization

Nucleoid‐associated protein HU, a conserved protein across eubacteria is necessary for maintaining the nucleoid organization and global regulation of gene expression. Mycobacterium tuberculosis HU (MtHU) is distinct from the other orthologues having 114 amino acid long carboxyl terminal extensions with a high degree of sequence similarity to eukaryotic histones. In this study, we demonstrate that the DNA binding property of MtHU is regulated by posttranslational modifications akin to eukaryotic histones. MtHU purified from M. tuberculosis cells is found to be acetylated on multiple lysine residues unlike the E. coli expressed recombinant protein. Using coimmunoprecipitation assay, we identified Eis as one of the acetyl transferases that interacts with MtHU and modifies it. Although Eis is known to acetylate aminoglycosides, the kinetics of acetylation showed that its protein acetylation activity on MtHU is robust. In vitro Eis modified MtHU at various lysine residues, primarily those located at the carboxyl terminal domain. Acetylation of MtHU caused reduced DNA interaction and alteration in DNA compaction ability of the NAP. Over‐expression of the Eis leads to hyperacetylation of HU and decompaction of genome. These results provide first insights into the modulation of the nucleoid structure by lysine acetylation in bacteria.     

Protective Effects of Triphala on Dermal Fibroblasts and Human Keratinocytes

Human skin is body’s vital organ constantly exposed to abiotic oxidative stress. This can have deleterious effects on skin such as darkening, skin damage, and aging. Plant-derived products having skin-protective effects are well-known traditionally. Triphala, a formulation of three fruit products, is one of the most important rasayana drugs used in Ayurveda. Several skin care products based on Triphala are available that claim its protective effects on facial skin. However, the skin protective effects of Triphala extract (TE) and its mechanistic action on skin cells have not been elucidated in vitro. Gallic acid, ellagic acid, and chebulinic acid were deduced by LC-MS as the major constituents of TE. The identified key compounds were docked with skin-related proteins to predict their binding affinity. The IC₅₀ values for TE on human dermal fibroblasts (HDF) and human keratinocytes (HaCaT) were 204.90 ± 7.6 and 239.13 ± 4.3 μg/mL respectively. The antioxidant capacity of TE was 481.33 ± 1.5 mM Trolox equivalents in HaCaT cells. Triphala extract inhibited hydrogen peroxide (H₂O₂) induced RBC haemolysis (IC₅₀ 64.95 μg/mL), nitric oxide production by 48.62 ± 2.2%, and showed high reducing power activity. TE also rescued HDF from H₂O₂-induced damage; inhibited H₂O₂ induced cellular senescence and protected HDF from DNA damage. TE increased collagen-I, involucrin and filaggrin synthesis by 70.72 ± 2.3%, 67.61 ± 2.1% and 51.91 ± 3.5% in HDF or HaCaT cells respectively. TE also exhibited anti-tyrosinase and melanin inhibition properties in a dose-dependent manner. TE increased the mRNA expression of collagen-I, elastin, superoxide dismutase (SOD-2), aquaporin-3 (AQP-3), filaggrin, involucrin, transglutaminase in HDF or HaCaT cells, and decreased the mRNA levels of tyrosinase in B16F10 cells. Thus, Triphala exhibits protective benefits on skin cells in vitro and can be used as a potential ingredient in skin care formulations.     

Isolation of a human DNA sequence which spans the fragile X

To identify the sequences involved in the expression of the fragile X and to characterize the molecular basis of the genetic lesion, we have constructed yeast artificial chromosomes (YACs) containing human DNA and have screened them with cloned DNA probes which map close to the fragile site at Xq27.3. We have isolated and partly characterized a YAC containing approximately 270 kb of human DNA from an X chromosome which expresses the fragile X. This sequence in a yeast artificial ring chromosome, XTY26, hybridizes to the two closest DNA markers, VK16 and Do33, which flank the fragile site. The human DNA sequence in XTY26 also spans the fragile site on chromosome in situ hybridization. When a restriction map of XTY26, derived by using infrequently cutting restriction enzymes, is compared with similar YAC maps derived from non-fragile-X patients, no large-scale differences are observed. This YAC, XTY26, may enable (a) the fragile site to be fully characterized at the molecular level and (b) the pathogenetic basis of the fragile-X syndrome to be determined.     

Comparisons of ruminal fermentation characteristics and microbial populations in bison and cattle

Ruminal microbial populations, fermentation characteristics, digestibility, and liquid flow rates in two ruminally cannulated bison and two ruminally cannulated Hereford steers fed a prairie hay diet were compared. No significant differences in anaerobic bacterial counts, volatile fatty acid concentrations, or ruminal pHs were evident between bison and cattle. Also, no significant differences in neutral detergent fiber digestibility, indigestible fiber retention time, or intake were detected between bison and cattle, although cattle had higher levels (P less than 0.08) of ruminal dry matter and indigestible fiber than bison. Bison had a smaller (P = .02) ruminoreticular volume, faster liquid dilution rates, and faster liquid turnover times than cattle. The average ruminal ammonia nitrogen concentration was higher (P = 0.02) in bison (1.17 mg/dl) than in cattle (0.79 mg/dl). Total ciliate protozoal counts and cell volume were greater (P = 0.07) in bison (32.8 x 10(4)/g and 407.1 x 10(-4) ml/g, respectively) than in cattle (15.7 x 10(4)/g and 162.2 x 10(-4) ml/g, respectively). Bison harbored higher (P less than 0.02) numbers of Dasytricha spp., Eudiplodinium maggii, Eudiplodinium bursa, and Epidinium spp. than cattle and possessed a type B protozoan population. The cattle possessed a mixed type A-type B population that was characterized by Ophryoscolex spp. and Polyplastron spp. in association with low concentrations of Epidinium spp. and Eudiplodinium maggii.     

Ca(2+)-mediated site-specific DNA cleavage and suppression of promiscuous activity of KpnI restriction endonuclease

The characteristic feature of type II restriction endonucleases (REases) is their exquisite sequence specificity and obligate Mg(2+) requirement for catalysis. Efficient cleavage of DNA only in the presence of Ca(2+) ions, comparable with that of Mg(2+), is previously not described. Most intriguingly, KpnI REase exhibits Ca(2+)-dependent specific DNA cleavage. Moreover, the enzyme is highly promiscuous in its cleavage pattern on plasmid DNAs in the presence of Mn(2+) or Mg(2+), with the complete suppression of promiscuous activity in the presence of Ca(2+). KpnI methyltransferase does not exhibit promiscuous activity unlike its cognate REase. The REase binds to oligonucleotides containing canonical and mapped noncanonical sites with comparable affinities. However, the extent of cleavage is varied depending on the metal ion and the sequence. The ability of the enzyme to be promiscuous or specific may reflect an evolutionary design. Based on the results, we suggest that the enzyme KpnI represents an REase evolving to attain higher sequence specificity from an ancient nonspecific nuclease.     

Transforming growth factor β receptor type 1 is essential for female reproductive tract integrity and function

The transforming growth factor β (TGFβ) superfamily proteins are principle regulators of numerous biological functions. Although recent studies have gained tremendous insights into this growth factor family in female reproduction, the functions of the receptors in vivo remain poorly defined. TGFβ type 1 receptor (TGFBR1), also known as activin receptor-like kinase 5, is the major type 1 receptor for TGFβ ligands. Tgfbr1 null mice die embryonically, precluding functional characterization of TGFBR1 postnatally. To study TGFBR1-mediated signaling in female reproduction, we generated a mouse model with conditional knockout (cKO) of Tgfbr1 in the female reproductive tract using anti-Müllerian hormone receptor type 2 promoter-driven Cre recombinase. We found that Tgfbr1 cKO females are sterile. However, unlike its role in growth differentiation factor 9 (GDF9) signaling in vitro, TGFBR1 seems to be dispensable for GDF9 signaling in vivo. Strikingly, we discovered that the Tgfbr1 cKO females develop oviductal diverticula, which impair embryo development and transit of embryos to the uterus. Molecular analysis further demonstrated the dysregulation of several cell differentiation and migration genes (e.g., Krt12, Ace2, and MyoR) that are potentially associated with female reproductive tract development. Moreover, defective smooth muscle development was also revealed in the uteri of the Tgfbr1 cKO mice. Thus, TGFBR1 is required for female reproductive tract integrity and function, and disruption of TGFBR1-mediated signaling leads to catastrophic structural and functional consequences in the oviduct and uterus.