Perinatal transmission of SHIV-SF162P3 in Macaca nemestrina

We developed a SHIV/macaque model of transmission from infected dams to their infants. Ten pregnant dams were infected intravenously with 100 MID(50) of macaque-titered SHIV-SF162P3 during the second trimester. Nine infants were born; the seven surviving beyond day of birth suckled for 6 months. Four of nine infants were infected (transmission rate = 44.4%), with one infection in utero, and three intrapartum and/or immediately post-birth via suckling. Varying levels of binding and neutralizing antibodies were transplacentally transferred to infants. Passive antibodies were detected in plasma on the day of birth and persisted for 5 weeks. Infants infected at or after birth controlled acute and post-acute viremia. Exposure to maternal SHIV-SF162P3 during birth and suckling in the presence of autologous maternal neutralizing antibodies may have affected transmission or pathogenesis in the infants. This transmission model can allow investigation of key parameters involved in perinatal transmission of HIV.     

HygR and PurR plasmid vectors for episomal transfection of Trypanosoma cruzi

This work describes the development and functional testing of two episomes for stable transfection of Trypanosoma cruzi. pHygD contained the 5'- and 3'- flanking regions of the gene encoding the cathepsin B-like protease of T. cruzi as functional trans-splicing and polyadenylation signals for the hygR ORF. Evidence is presented to support extrachromosomal maintenance and organization as tandem repeats in transfected parasites. pPac was derived from pHygD by replacement of the entire hygR ORF with a purR coding region. The ability to modify pHygD and the availability of the complete DNA sequence make these plasmids useful tools for the genetic manipulation of T. cruzi.     

Pathogenesis of acute ulceration response (AUR) in hybrid striped bass

In a previous study, we discovered that acute confinement stress causes rapid ulceration of the fins of hybrid striped bass Morone saxatilis female x M. chrysops male (Noga et al. 1998. Vet Pathol 35:102-107). In this paper, we report the development of a reproducible model for studying this phenomenon in juvenile hybrid striped bass. We also determined how quickly ulceration could develop in acutely stressed fish and documented the sequential light microscopic and ultrastructural changes associated with this response. When hybrid striped bass were subjected to a standardized confinement protocol, the pathological response was extremely rapid (fin ulceration began to develop within 15 min of confinement). Grossly, the distal edges of the fins became blanched, and melanophores aggregated near the basement membrane and dermis after 15 min of confinement. Microscopically, the earliest detectable change in the fins, which occurred within 15 min of confinement, was swelling and loss of microridges of the outermost epidermal cells; this was followed by epidermal erosion. After 30 min of stress, epidermal ulceration developed at the distal edges of the fins. At this time, both necrotic and apoptotic epidermal cells were present. The middle and basal epidermal layers were severely spongiotic and the dermis and hypodermis were edematous. Over longer periods (up to 2 h), lesions were similar but increasingly more severe, progressing from the distal edge of the fin towards the base. The response to acute stress showed a significant correlation between confinement period and severity of the pathological changes (epidermal degeneration, epidermal ulceration and leukocyte infiltration). Also, we demonstrated that epidermal damage was not restricted to the fins but also affected the body skin and eyes. The ventral area of the body and the corneal epithelium of stressed fish were ulcerated; however, skin on the head and operculum was not affected, suggesting a site-specific mode of damage. In stressed fish, epidermal ulceration was found in 67 to 97% of all fins, 88% of skin on the ventrum, and 67% of corneas, while control fish had only very mild epidermal ulceration in the few fish in which it was present (on 5 to 10% of the fins, but not on the ventral skin or corneas). Due to the widespread damage to epidermal tissues of the body surface, we have named this the acute ulceration response (AUR). Our study indicates that acute confinement can rapidly cause significant damage to epidermal and ocular epithelium. AUR might be a primary cause of morbidity in acutely stressed fish.     

Role of nitric oxide in hepatic microvascular injury elicited by acetaminophen in mice

Nitric oxide (NO) is suggested to play a role in liver injury elicited by acetaminophen (APAP). Hepatic microcirculatory dysfunction also is reported to contribute to the development of the injury. As a result, the role of NO in hepatic microcirculatory alterations in response to APAP was examined in mice by in vivo microscopy. A selective inducible NO synthase (iNOS) inhibitor,l-N6-(1-iminoethyl)-lysine (L-NIL), or a nonselective NOS inhibitor, NG-nitro-l-arginine methyl ester (L-NAME), was intraperitoneally administered to animals 10 min before APAP gavage. L-NIL suppressed raised alanine aminotransferase (ALT) values 6 h after APAP, whereas L-NAME increased those 1.7-fold. Increased ALT levels were associated with hepatic expression of iNOS. L-NIL, but not L-NAME, reduced the expression. APAP caused a reduction (20%) in the numbers of perfused sinusoids. L-NIL restored the sinusoidal perfusion, but L-NAME was ineffective. APAP increased the area occupied by infiltrated erythrocytes into the extrasinusoidal space. L-NIL tended to minimize this infiltration, whereas L-NAME further enhanced it. APAP caused an increase (1.5-fold) in Kupffer cell phagocytic activity. This activity in response to APAP was blunted by L-NIL, whereas L-NAME further elevated it. L-NIL suppressed APAP-induced decreases in hepatic glutathione levels. These results suggest that NO derived from iNOS contributes to APAP-induced parenchymal cell injury and hepatic microcirculatory disturbances. L-NIL exerts preventive effects on the liver injury partly by inhibiting APAP bioactivation. In contrast, NO derived from constitutive isoforms of NOS exerts a protective role in liver microcirculation against APAP intoxication and thereby minimizes liver injury.     

Structure of uPAR, plasminogen, and sugar-binding sites of the 300 kDa mannose 6-phosphate receptor

The 300 kDa cation-independent mannose 6-phosphate receptor (CI-MPR) mediates the intracellular transport of newly synthesized lysosomal enzymes containing mannose 6-phosphate on their N-linked oligosaccharides. In addition to its role in lysosome biogenesis, the CI-MPR interacts with a number of different extracellular ligands at the cell surface, including latent transforming growth factor-beta, insulin-like growth factor-II, plasminogen, and urokinase-type plasminogen activator receptor (uPAR), to regulate cell growth and motility. We have solved the crystal structure of the N-terminal 432 residues of the CI-MPR at 1.8 A resolution, which encompass three out of the 15 repetitive domains of its extracytoplasmic region. The three domains, which exhibit similar topology to each other and to the 46 kDa cation-dependent mannose 6-phosphate receptor, assemble into a compact structure with the uPAR/plasminogen and the carbohydrate-binding sites situated on opposite faces of the molecule. Knowledge of the arrangement of these three domains has allowed us to propose a model of the entire extracytoplasmic region of the CI-MPR that provides a context with which to envision the numerous binding interactions carried out by this multi-faceted receptor.     

4-Amino cyclohexylglycine analogues as potent dipeptidyl peptidase IV inhibitors

Substituted 4-amino cyclohexylglycine analogues were evaluated for DP-IV inhibitory properties. Bis-sulfonamide 15e was an extremely potent 2.6 nM inhibitor of the enzyme with excellent selectivity over all counterscreens. 2,4-difluorobenzenesulfonamide 15b and 1-naphthyl amide 16b, however, combined an acceptable in vitro profile with good pharmacokinetic properties in the rat, and 15b was orally efficacious at 3 mpk in an OGTT in lean mice.     

Benzimidazolone p38 inhibitors

The synthesis and in vitro p38 alpha activity of a novel series of benzimidazolone inhibitors is described. The p38 alpha SAR is consistent with a mode of binding wherein the benzimidazolone carbonyl serves as the H-bond acceptor to Met109 of p38 alpha in a manner analogous to the pyridine nitrogen of prototypical pyridylimidazole p38 inhibitors. Potent p38 alpha activity comparable to that of several previously reported p38 inhibitors is observed for this novel chemotype.