Development of a yeast-based recombination cloning/system for the analysis of gene products from diverse human immunodeficiency virus type 1 isolates

A recent shift from studies on a few subtype B laboratory human immunodeficiency virus type 1 (HIV-1) clones to analyses of extremely diverse primary HIV-1 isolates from different subtype requires the development of a rapid and generic cloning technique. This report describes the use of gap repair/recombination in yeast to shuttle env, gag, and pol genes from diverse HIV-1 subtypes into a DNA vector that can be amplified in bacteria and can express the gene of interest in mammalian cells. These diverse HIV-1 genes have also been introduced into an infectious clone to produce chimeric viruses that are useful for studies on drug susceptibility, receptor binding and fitness.     

Intestinal uptake of quercetin-3-glucoside in rats involves hydrolysis by lactase phlorizin hydrolase

Quercetin has antioxidant, anti-inflammatory, antiproliferative and anticarcinogenic properties. In plant foods, quercetin occurs mainly bound to various sugars via a beta-glycosidic link. We hypothesized that lactase phlorizin hydrolase (LPH), an enzyme at the brush border membrane of intestinal cells, is involved in the in vivo intestinal uptake of quercetin-sugars. To study this, we measured the appearance of quercetin metabolites in plasma and perfusate after perfusing the jejunum and ileum with 50 micro mol/L quercetin-3-glucoside in an in situ rat perfusion model. LPH was inhibited by the selective LPH inhibitor N-butyldeoxygalactonojirimycin (0, 0.5, 2 or 10 mmol/L) (n = 5 rats/group). Quercetin in plasma and perfusion buffer was determined by HPLC with CoulArray detection. Results are given as means +/- SEM. In the perfusion buffer, 13.8 +/- 0.7 micro mol/L quercetin-3-glucoside was hydrolyzed during intestinal passage. Co-perfusion with 0.5, 2 and 10 mmol/L N-butyldeoxygalactonojirimycin resulted in 38% (P < 0.05), 50% (P < 0.01) and 67% (P < 0.01) less hydrolysis, respectively. Plasma concentrations of quercetin in the corresponding groups were 36% (P = 0.12), 55% (P < 0.01) and 75% (P < 0.01) lower than in controls (1.23 +/- 0.22 micro mol/L). These data suggest that LPH is a major determinant of intestinal absorption of quercetin-3-glucoside in rats.     

Cochlin Isoforms and Their Interaction with CTL2 (SLC44A2) in the Inner Ear

Choline transporter-like protein 2 (CTL2) is a multi-transmembrane protein expressed on inner ear supporting cells that was discovered as a target of antibody-induced hearing loss. Its function is unknown. A 64 kDa band that consistently co-precipitates with CTL2 from inner ear extracts was identified by mass spectroscopy as cochlin. Cochlin is an abundant inner ear protein expressed as multiple isoforms. Its function is also unknown, but it is suspected to be an extracellular matrix component. Cochlin is mutated in individuals with DFNA9 hearing loss. To investigate the CTL2–cochlin interaction, antibodies were raised to a cochlin-specific peptide. The antibodies identify several cochlin polypeptides on western blots and are specific for cochlin. We show that the heterogeneity of the cochlin isoforms is caused, in part, by in vivo post-translational modification by N-glycosylation and, in part, caused by alternative splicing. We verified that antibody to CTL2 co-immunoprecipitates cochlin from the inner ear and antibody to cochlin co-immunoprecipitates CTL2. Using cochlear cross-sections, we show that CTL2 is more widely distributed than previously described, and its prominent expression on cells facing the scala media suggests a possible role in homeostasis. A prominent but previously unreported ribbon-like pattern of cochlin in the basilar membrane was demonstrated, suggesting an important role for cochlin in the structure of the basilar membrane. CTL2 and cochlin are expressed in close proximity in the inner sulcus, the spiral prominence, vessels, limbus, and spiral ligament. The possible functional significance of CTL2–cochlin interactions remains unknown.     

Minimizing wound complications in cochlear implant surgery.

OBJECTIVE: An extended postauricular incision has replaced the standard C-shaped scalp flap for cochlear implant surgery at our institution. The postoperative wound complication rates of the two incisions were evaluated. STUDY DESIGN: This study was a retrospective case review. SETTING: This study was performed in a tertiary referral center. PATIENTS: A total of 256 adult and pediatric patients who underwent cochlear implantation during a 10-year period (1986 to 1996) were reviewed. MAIN OUTCOME MEASURE: Postoperative wound complications were identified. Major complications included flap necrosis, wound dehiscence with or without implant exposure, and wound infection requiring hospitalization. Hematoma, seroma, or superficial wound infections were considered minor complications. RESULTS: There were 6 major and 6 minor complications among 116 patients with the standard scalp flap (complication rate, 10.3%). There was only 1 minor complication among 140 implants using the postauricular incision (0.7%). CONCLUSION: The extended postauricular incision appears to significantly reduce the incidence of wound complications in cochlear implant surgery.     

Prostatic evaluation by transrectal sonography with histopathologic correlation: the echopenic appearance of early carcinoma

Fifty-two patients with clinical stage A and B carcinomas of the prostate were imaged by ultrasound (US) transrectally with a 5-MHz linear array transducer and transabdominally with a 3-MHz sector scanner prior to radical prostatectomy. The fresh specimens of 44 prostate glands were scanned in a water bath with a 5-MHz linear array transducer in multiple planes. In all cases, histopathologic correlation was obtained. Prostatic carcinoma presented as an echopenic lesion in 54% of the specimens, as a slightly hypoechoic area in 22%, and could not be identified in 24% because of its isoechoic characteristics. In contrast to many previous reports, no instance of echogenic cancer was observed.