Background: Ceramide is an important second messenger that has diverse cellular and biological effect. It is a specific and potent inducer of apoptosis and suppressor of cell growth. In leukemia, chemoresistance generally developed due to deregulated ceramide metabolism. In combinatorial treatment strategies of leukemia, few components have the capability to increases ceramide production. Manipulation in ceramide production by physiological and pharmacological modulators therefore will give additive effect in leukemia chemotherapy. Results: Here, we show that Withanolide D (C ₄ β-C ₅ β,C ₆ β-epoxy-1-oxo-,20β, dihydroxy-20 S ,22 R -witha-2,24-dienolide; WithaD), a pure herbal compound isolated from Withania somnifera could effectively induces apoptosis in a dose and time dependant manner both in myeloid (K562) and lymphoid (MOLT-4) cells being nontoxic to normal lymphocytes and control proliferative cells. WithaD potentially augment ceramide production in these cells. Downstream of ceramide, WithaD acted on MKK group of proteins and significantly increased JNK and p38MAPK phosphorylation. Pharmacological inhibition of p38MAPK and JNK proves their cooperative action on WithaD-induced cell death. Dissecting the cause of ceramide production, we found activation of neutral sphingomyelinase and showed neutral-sphingomyelinase 2 (N-SMase 2) is a critical mediator of WithaD-induced apoptosis. Knockdown of N-SMase 2 by siRNA and inhibitor of N-SMase (GW4869) significantly reduced WithaD-induced ceramide generation and phosphorylation of MKK4 and MKK3/6, whereas phosphorylation of MKK7 was moderately regulated in leukemic cells. Also, both by silencing of N-SMase 2 and/or blocking by GW4869 protects these cells from WithaD-mediated death and suppressed apoptosis, whereas Fumonisin B1, an inhibitor of ceramide synthase, did not have any effect. Additionally, WithaD effectively induced apoptosis in freshly isolated lymphoblasts from patients and the potent cell killing activity was through JNK and p38MAPK activation. Conclusion: Our results demonstrate that WithaD enhance the ceramide accumulation by activating N-SMase 2, modulate phosphorylation of the JNK and p38MAPK and induced apoptosis in both myeloid and lymphoid cells along with primary cells derived from leukemia patients. Taken together, this pure herbal compound (WithaD) may consider as a potential alternative tool with additive effects in conjunction with traditional chemotherapeutic treatment, thereby accelerate the process of conventional drug development.

Leishmaniasis remains a major health problem of the tropical and subtropical world. The visceral form causes the most fatalities if left untreated. Dramatic increases in the rates of infection and drug resistance and the non-availability of safe vaccines have highlighted the need for identification of novel and inexpensive anti-leishmanial agents. This study reports that racemoside A, a water-soluble steroidal saponin purified from the fruits of Asparagus racemosus, is a potent anti-leishmanial molecule effective against antimonial-sensitive (strain AG83) and -unresponsive (strain GE1F8R) Leishmania donovani promastigotes, with IC₅₀ values of 1.15 and 1.31 μg ml⁻¹, respectively. Incubation of promastigotes with racemoside A caused morphological alterations including cell shrinkage, an aflagellated ovoid shape and chromatin condensation. This compound exerts its leishmanicidal effect through the induction of programmed cell death mediated by the loss of plasma membrane integrity as detected by binding of annexin V and propidium iodide, loss of mitochondrial membrane potential culminating in cell-cycle arrest at the sub-G₀/G₁ phase, and DNA nicking shown by deoxynucleotidyltransferase-mediated dUTP end labelling (TUNEL). Racemoside A also showed significant activity against intracellular amastigotes of AG83 and GE1F8R at a 7–8-fold lower dose, with IC₅₀ values of 0.17 and 0.16 μg ml⁻¹, respectively, and was non-toxic to murine peritoneal macrophages up to a concentration of 10 μg ml⁻¹. Hence, racemoside A is a potent anti-leishmanial agent that merits further pharmacological investigation.

The opportunism of Pseudomonas aeruginosa (PA) in immunocompromised hosts prompted us to explore the potential role of sialic acids (Sia) in this phenomenon. Culture of PA in the presence of exogenous Sia resulted in linkage‐specific incorporation of Sia which was associated with decreased complement deposition on the bacteria. Sia acquired by PA mediated enhanced binding of bacteria to recombinant‐CHO cells expressing human siglec‐7 or siglec‐9, as well as to human NK‐cells and monocytes naturally expressing these siglecs. Therefore, Sia may be acquired by PA in the host and contribute to bacterial pathogenicity and host‐cell interactions via reduction of complement deposition and siglec‐dependent recognition.

As an acute-phase protein, human C-reactive protein (CRP) is clinically important. CRPs were purified from several samples in six different pathological conditions, where their levels ranged from 22 to 342 μg/ml. Small, but significant, variations in electrophoretic mobilities on native PAGE suggested differences in molecular mass, charge and/or shape. Following separation by SDS/PAGE, they showed single subunits with some differences in their molecular masses ranging between 27 and 30.5 kDa, but for a particular disease, the mobility was the same for CRPs purified from multiple individuals or pooled sera. Isoelectric focusing (IEF) also indicated that the purified CRPs differed from each other. Glycosylation was demonstrated in these purified CRPs by Digoxigenin kits, neuraminidase treatment and binding with lectins. The presence of N-linked sugar moiety was confirmed by N-glycosidase F digestion. The presence of sialic acid, glucose, galactose and mannose has been demonstrated by gas liquid chromatography, mass spectroscopic and fluorimetric analysis. Matrix-assisted laser-desorption ionization analysis of the tryptic digests of three CRPs showed systematic absence of two peptide fragments, one at the N-terminus and the other near the C-terminus. Model-building suggested that the loss of these fragments exposed two potential glycosylation sites on a cleft floor keeping the protein–protein interactions in pentraxins and calcium-dependent phosphorylcholine-binding qualitatively unaffected. Thus we have convincingly demonstrated that human CRP is glycosylated in some pathological conditions.