Activation-induced nonresponsiveness: a Th-dependent regulatory checkpoint in the CTL response.

CD8 T cells undergo autocrine IL-2-dependent proliferation upon TCR engagement and costimulation, but within 3-4 days, they become activation-induced nonresponsive (AINR) and display a split anergy. They can lyse targets and secrete IFN-gamma but they cannot produce IL-2 in response to TCR ligation and costimulation, due at least in part to an inability to up-regulate mitogen-activated protein kinases and IL-2 mRNA. Exogenous IL-2 can drive continued proliferation of AINR cells and nonresponsiveness is reversed within 1-2 days so that Ag-driven proliferation can resume. Mitogen-activated protein kinases and IL-2 mRNA can again be up-regulated, but "rewiring" has occurred so that these events no longer depend upon costimulation; TCR engagement is sufficient. Development of AINR appears to be a normal part of the differentiation program of CD8 T cells, providing a regulatory checkpoint to convert the initial helper-independent response to one that depends upon CD4 T cell help for continued expansion of the effector CTL. Once permission is given, in the form of IL-2, to pass this checkpoint, the CTL can make a prolonged response to persisting Ag in the absence of further CD4 T cell help.     

Dynamics of CCR5 expression by CD4(+) T cells in lymphoid tissues during simian immunodeficiency virus infection

Early viral replication and profound CD4(+) T-cell depletion occur preferentially in intestinal tissues of macaques infected with simian immunodeficiency virus (SIV). Here we show that a much higher percentage of CD4(+) T cells in the intestine express CCR5 compared with those found in the peripheral blood, spleen, or lymph nodes. In addition, the selectivity and extent of the CD4(+) T-cell loss in SIV infection may depend upon these cells coexpressing CCR5 and having a "memory" phenotype (CD45RA(-)). Following intravenous infection with SIVmac251, memory CD4(+) CCR5(+) T cells were selectively eliminated within 14 days in all major lymphoid tissues (intestine, spleen, and lymph nodes). However, the effect on CD4(+) T-cell numbers was most profound in the intestine, where cells of this phenotype predominate. The CD4(+) T cells that remain after 14 days of infection lacked CCR5 and/or were naive (CD45RA(+)). Furthermore, when animals in the terminal stages of SIV infection (with AIDS) were examined, virtually no CCR5-expressing CD4(+) T cells were found in lymphoid tissues, and all of the remaining CD4(+) T cells were naive and coexpressed CXCR4. These findings suggest that chemokine receptor usage determines which cells are targeted for SIV infection and elimination in vivo.     

DNA fingerprinting of Peronospora parasitica,a biotrophic fungal pathogen of crucifers

The fungus Peronospora parasitica (Pers. ex Fr.) Fr. is an obligate biotroph infecting a wide range of host species in the family Cruciferae. Isolates from different hosts are morphologically similar, and pathotypes are usually distinguished on the basis of host range. Random Amplified Polymorphic DNA (RAPD) fingerprints were generated from a range of P. parasitica isolates from different Brassica species. Reaction conditions, in particular DNA template, primer and Mg²⁺ concentrations, were optimized to ensure that amplifications were reproducible. Possible artefacts arising through host plant DNA were assessed by including such DNA in control reactions. Confirmation that diagnostic RAPD bands were generated from fungal DNA was also obtained by Southern hybridization of a RAPD band to genomic fungal DNA. By screening 20 decamer primers, 2 were found to detect sufficient genetic variation to allow complete differentiation between pathotypes. These results illustrate the potential value of RAPDs for detecting polymorphisms between isolates of a non-culturable plant pathogenic fungus.     

Molecular characterization of the P1-like adhesin gene from Mycoplasma pirum.

A DNA fragment has been isolated from the genome of Mycoplasma pirum by use of a genetic probe derived from the conserved region within the genes for the major adhesins of Mycoplasma genitalium and Mycoplasma pneumoniae. A gene encoding an adhesin-like polypeptide was localized, and sequence analysis indicated a G + C content of only 28%, with A- and T-rich codons being preferentially used. A total of 91% of positions 3 were either A or T. The deduced polypeptide is 1,144 amino acids long (126 kDa) and shows 26% identity with the adhesins of M. genitalium and M. pneumoniae. Other features in common with these two membrane proteins include a similar hydropathic profile and a proline-rich C terminus. Antibodies were prepared by using as an immunogen a peptide derived from the C terminus of the M. pirum adhesin-like polypeptide and were found to recognize on immunoblots a 126-kDa polypeptide from an M. pirum cellular extract. The characterization of the adhesin-like gene is a first step toward a better understanding of the mechanisms allowing this human mycoplasma to attach to host cells.     

Bioprocess model building using artificial neural networks

Artificial neural networks are made upon of highly interconnected layers of simple neuron-like nodes. The neurons act as non-linear processing elements within the network. An attractive property of artificial neural networks is that given the appropriate network topology, they are capable of learning and characterising non-linear functional relationships. Furthermore, the structure of the resulting neural network based process model may be considered generic, in the sense that little prior process knowledge is required in its determination. The methodology therefore provides a cost efficient and reliable process modelling technique. One area where such a technique could be useful is biotechnological systems. Here, for example, the use of a process model within an estimation scheme has long been considered an effective means of overcoming inherent on-line measurement problems. However, the development of an accurate process model is extremely time consuming and often results in a model of limited applicability. Artificial neural networks could therefore prove to be a useful model building tool when striving to improve bioprocess operability. Two large scale industrial fermentation systems have been considered as test cases; a fed-batch penicillin fermentation and a continuous mycelial fermentation. Both systems serve to demonstrate the utility, flexibility and potential of the artificial neural network approach to process modelling.     

Design and synthesis of novel thiadiazole-thiazolone hybrids as potential inhibitors of the human mitotic kinesin Eg5

A novel series of 1,3,4-thiadiazole-thiazolone hybrids 5a–v were designed, synthesized, characterized, and evaluated against the basal and the microtubule (MT)-stimulated ATPase activity of Eg5. From the evaluated derivatives, 5h displayed the highest inhibition with an IC₅₀ value of 13.2?µM against the MT-stimulated Eg5 ATPase activity. Similarly, compounds 5f and 5i also presented encouraging inhibition with IC₅₀ of 17.2?µM and 20.2?µM, respectively. A brief structure–activity relationship (SAR) analysis indicated that 2-chloro and 4-nitro substituents on the phenyl ring of the thiazolone motif contributed significantly to enzyme inhibition. An in silico molecular docking study using the crystal structure of Eg5 further supported the SAR and reasoned the importance of crucial molecular protein–ligand interactions in influencing the inhibition of the ATPase activity of Eg5. The magnitude of the electron-withdrawing functionalities over the hybrids and the critical molecular interactions contributed towards higher in vitro potency of the compounds. The drug-like properties of the synthesized compounds 5a–v were also calculated based on the Lipinski’s rule of five and in silico computation of key pharmacokinetic parameters (ADME). Thus, the present work unveils these hybrid molecules as novel Eg5 inhibitors with promising drug-like properties for future development.