Chlamydia trachomatis Infection of Endocervical Epithelial Cells Enhances Early HIV Transmission Events

Chlamydia trachomatis causes a predominantly asymptomatic, but generally inflammatory, genital infection that is associated with an increased risk for HIV acquisition. Endocervical epithelial cells provide the major niche for this obligate intracellular bacterium in women, and the endocervix is also a tissue in which HIV transmission can occur. The mechanism by which CT infection enhances HIV susceptibility at this site, however, is not well understood. Utilizing the A2EN immortalized endocervical epithelial cell line grown on cell culture inserts, we evaluated the direct role that CT-infected epithelial cells play in facilitating HIV transmission events. We determined that CT infection significantly enhanced the apical-to-basolateral migration of cell-associated, but not cell-free, HIV_BaL, a CCR5-tropic strain of virus, across the endocervical epithelial barrier. We also established that basolateral supernatants from CT-infected A2EN cells significantly enhanced HIV replication in peripheral mononuclear cells and a CCR5+ T cell line. These results suggest that CT infection of endocervical epithelial cells could facilitate both HIV crossing the mucosal barrier and subsequent infection or replication in underlying target cells. Our studies provide a mechanism by which this common STI could potentially promote the establishment of founder virus populations and the maintenance of local HIV reservoirs in the endocervix. Development of an HIV/STI co-infection model also provides a tool to further explore the role of other sexually transmitted infections in enhancing HIV acquisition.     

Fragment of the gene encoding chymotrypsin and trypsin inhibitor protein of potato tubers

Product of polymerase chain reaction designated as PKPIJ-B was isolated after amplification from genomic DNA of potato (Solarium tuberosum L., Zhukov Jubilee cultivar) using the designed primers. Nucleotide sequence of PKPIJ-B was determined and amino acid sequence of protein was restored. Analysis of this sequence has allowed us to suggest that isolated gene fragment encodes chymotrypsin and trypsin inhibitor protein (PKCI-23 potato Kunitz-type chymotrypsin inhibitor) of potato tubers.     

Intracellular Calcium Deficits in Drosophila Cholinergic Neurons Expressing Wild Type or FAD-Mutant Presenilin

Much of our current understanding about neurodegenerative diseases can be attributed to the study of inherited forms of these disorders. For example, mutations in the presenilin 1 and 2 genes have been linked to early onset familial forms of Alzheimer''s disease (FAD). Using the Drosophila central nervous system as a model we have investigated the role of presenilin in one of the earliest cellular defects associated with Alzheimer''s disease, intracellular calcium deregulation. We show that expression of either wild type or FAD-mutant presenilin in Drosophila CNS neurons has no impact on resting calcium levels but does give rise to deficits in intracellular calcium stores. Furthermore, we show that a loss-of-function mutation in calmodulin, a key regulator of intracellular calcium, can suppress presenilin-induced deficits in calcium stores. Our data support a model whereby presenilin plays a role in regulating intracellular calcium stores and demonstrate that Drosophila can be used to study the link between presenilin and calcium deregulation.     

Structural analysis of a non-contiguous second-site revertant in T4 lysozyme shows that increasing the rigidity of a protein can enhance its stability.

The mutation Glu108-->Val (E108V) in T4 lysozyme was previously isolated as a second-site revertant that specifically compensated for the loss of function associated with the destabilizing substitution Leu99-->Gly (L99G). Surprisingly, the two sites are 11 A apart, with Leu99 in the core and Glu108 on the surface of the protein. In order to better understand this result we have carried out a detailed thermodynamic, enzymatic and structural analysis of these mutant lysozymes as well as a related variant with the substitution Leu99-->Ala. It was found that E108V does increase the stability of L99G, but it also increases the stability of both the wild-type protein and L99A by essentially equal amounts. The effects of E108V on enzymatic activity are more complicated. The mutation slightly reduces the maximal rate of cell wall hydrolysis of wild-type, L99G and L99A. At the same time, L99G is an unstable protein and rapidly loses activity during the course of the assay, especially at temperatures above 20 degrees C. Thus, even though the double mutant L99G/E108V has a slightly lower maximal rate than L99G, over a period of 20-30 minutes it hydrolyzes more substrate. This decrease in the rate of thermal inactivation appears to be the basis of the action of E108V as a second-site revertant of L99G. Mutant L99A creates a cavity of volume 149 A(3). Instead of enlarging this cavity, mutant L99G results in a 4-5 A displacement of part of helix F (residues 108-113), creating a solvent-accessible declivity. In the double mutant, L99G/E108V, this helix returns to a position akin to wild-type, resulting in a cavity of volume 203 A(3). Whether the mutation Glu108-->Val is incorporated into either wild-type lysozyme, or L99A or L99G, it results in a decrease in crystallographic thermal factors, especially in the helices that include residues 99 and 108. This increase in rigidity, which appears to be due to a combination of increased hydrophobic stabilization plus a restriction of conformational fluctuation, provides a structural basis for the increase in thermostability.     

Assay of reducing sugars in the nanomole range with 2,2'-bicinchoninate

The photometric assay of reducing sugars with 2,2'-bicinchoninate was improved and its conditions were optimized. Optical density is linear between 1 and 25 nmol sugar/sample, and the assay is not affected by borate, phosphate, or other buffer anions. The molar extinction coefficients produced by the standard procedure with the 12 most common monosaccharides occurring in carbohydrates and conjugated glycocompounds are listed.