Cloning of a human UDP-galactose:2-acetamido-2-deoxy-D-glucose 3beta-galactosyltransferase catalyzing the formation of type 1 chains

Biochemical evidence suggests that the galactosyltransferase activity synthesizing type 1 carbohydrate chains is separate from the well characterized enzyme that is responsible for the synthesis of type 2 chains. This was recently confirmed by the cloning, from melanoma cells, of an enzyme capable of synthesizing type 1 chains, which was shown to have no homology to other galactosyltransferases. We report here the molecular cloning and functional expression of a second human beta3-galactosyltransferase distinct from the melanoma enzyme. The new beta3-galactosyltransferase has homology to the melanoma enzyme in the putative catalytic domain, but has longer cytoplasmic and stem regions and a carboxyl-terminal extension. Northern blots showed that the new gene is present primarily in brain and heart. When transfected into mammalian cells, this gene directs the synthesis of type 1 chains as determined by a monoclonal antibody specific for sialyl Lewisa. A soluble version of the cloned enzyme was expressed in insect cells and purified. The soluble enzyme readily catalyzes the transfer of galactose to GlcNAc to form Gal(beta1-3)GlcNAc. It also has a minor but distinct transfer activity toward Gal, LacNAc, and lactose, but is inactive toward GalNAc.     

Reference values for height and weight in Prader-Willi syndrome based on 315 patients

The spontaneous growth of 315 patients (109 girls and 208 boys) with Prader-Willi syndrome (PWS) was analysed in a mixed longitudinal and cross-sectional manner. 33 patients were seen in the department between 1970 and 1994; height and weight of 76 patients from Germany were evaluated by means of a questionnaire with detailed measuring instructions, and 206 definite cases were added from the literature. Mean ( SD) length of newborn babies with PWS was 50.2+/-2.8 cm (145 boys) and 48.9 3.3 cm (79 girls). Mean weight at birth was 2945 570 g in boys and 2782+/-594 g in girls. During the 1st year, the children's growth was nearly normal, thereafter short stature was present in approximately 50% of PWS patients. Between 3 and 13 years of age, the 50th percentile for height in PWS is roughly identical with the 3rd percentile in healthy controls. Body weight was normal for all boys and girls during the first 2 years. Thereafter, a rapid weight gain occurred; after an age of 10 years weight-for-height index in nearly all patients exceeded the normal range. The extent of pubertal growth was reduced for the group. Mean adult height was 161.6+/-8.1 cm (23 males) and 150.2+/-5.5 cm (21 females). Head circumference for age was normal for boys and girls. CONCLUSION: Reference data on spontaneous development of growth and weight gain of children with Prader-Willi syndrome are described allowing a better counselling of patients and parents.     

Apoptosis after traumatic human spinal cord injury

OBJECT: Apoptosis is a form of programmed cell death seen in a variety of developmental and disease states, including traumatic injuries. The main objective of this study was to determine whether apoptosis is observed after human spinal cord injury (SCI). The spatial and temporal expression of apoptotic cells as well as the nature of the cells involved in programmed cell death were also investigated. METHODS: The authors examined the spinal cords of 15 patients who died between 3 hours and 2 months after a traumatic SCI. Apoptotic cells were found at the edges of the lesion epicenter and in the adjacent white matter, particularly in the ascending tracts, by using histological (cresyl violet, hematoxylin and eosin) and nuclear staining (Hoechst 33342). The presence of apoptotic cells was supported by staining with the terminal deoxynucleotidyl transferase-mediated deoxyuridinetriphosphate nick-end labeling technique and confirmed by immunostaining for the processed form of caspase-3 (CPP-32), a member of the interleukin-1beta-converting enzyme/Caenorhabditis elegans D 3 (ICE/CED-3) family of proteases that plays an essential role in programmed cell death. Apoptosis in this series of human SCIs was a prominent pathological finding in 14 of the 15 spinal cords examined when compared with five uninjured control spinal cords. To determine the type of cells undergoing apoptosis, the authors immunostained specimens with a variety of antibodies, including glial fibrillary acidic protein, 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNPase), and CD45/68. Oligodendrocytes stained with CNPase and a number of apoptotic nuclei colocalized with positive staining for this antibody. CONCLUSIONS: These results support the hypothesis that apoptosis occurs in human SCIs and is accompanied by the activation of caspase-3 of the cysteine protease family. This mechanism of cell death contributes to the secondary injury processes seen after human SCI and may have important clinical implications for the further development of protease inhibitors to prevent programmed cell death.     

Tonsillar carcinoma: analysis of treatment results

A diffusion cell with an artificial membrane and the single-pass perfused rabbit ear were used to evaluate the percutaneous absorption of clonazepam from various 2-hydroxyethyl acetate (HEA) patches. The influence on drug permeation of the various type of enhancers (isopropylmyristate, lauryl alcohol, propylene glycol and water) in the patches was tested. A comparison between the two types of systems of percutaneous absorption of clonazepam has been done. The results showed that HEA patches produce controlled uniform drug release, modulated by the addition of enhancers.     

Mucosal immunity to herpes simplex virus type 2 infection in the mouse vagina is impaired by in vivo depletion of T lymphocytes

Intravaginal (IVAG) inoculation of wild-type herpes simplex virus type 2 (HSV-2) in mice causes epithelial infection followed by lethal neurological illness, while IVAG inoculation of attenuated HSV-2 causes epithelial infection followed by development of protective immunity against subsequent IVAG challenge with wild-type virus. The role of T cells in this immunity was studied by in vivo depletion of these cells with monoclonal antibodies. Three groups of mice were used for each experiment: nonimmune/challenged mice, immune/challenged mice, and immune depleted mice [immune mice depleted of a T-cell subset(s) shortly before challenge with HSV-2]. Mice were assessed for epithelial infection 24 h after challenge, virus protein in the vaginal lumen 3 days after challenge, and neurological illness 8 to 14 days after challenge. Monoclonal antibodies to CD4, CD8, or Thy-1 markedly reduced T cells in blood, spleen, and vagina, but major histocompatibility complex class II antigens were still partially upregulated in the vaginal epithelium after virus challenge, indicating that virus-specific memory T-cell function was not entirely eliminated from the vagina. Nevertheless, immune mice depleted of CD4+ and CD8+ T cells, Thy-1+ T cells, or CD8+ T cells alone had greater viral infection in the vaginal epithelium than nondepleted immune mice, indicating that T cells contribute to immunity against vaginal HSV-2 infection. All immune depleted mice retained substantial immunity to epithelial infection and were immune to neurological illness, suggesting that other immune mechanisms such as virus-specific antibody may also contribute to immunity.     

Lepromatous uveitis diagnosed by iris biopsy

Ocular leprosy is rarely seen in developed countries. We report the long-term follow-up of a patient with bilateral uveitis, glaucoma, and keratitis. Skin, iris and aqueous humor biopsies disclosed abundant Wade-Fite-positive organisms consistent with Mycobacterium leprae. Leprosy must be considered in the differential diagnosis of keratitis and uveitis.     

Characterization of recombinant soybean leghemoglobin a and apolar distal histidine mutants

The cDNA for soybean leghemoglobin a (Lba) was cloned from a root nodule cDNA library and expressed in Escherichia coli. The crystal structure of the ferric acetate complex of recombinant wild-type Lba was determined at a resolution of 2.2 A. Rate constants for O2, CO and NO binding to recombinant Lba are identical with those of native soybean Lba. Rate constants for hemin dissociation and auto-oxidation of wild-type Lba were compared with those of sperm whale myoglobin. At 37 degrees C and pH 7, soybean Lba is much less stable than sperm whale myoglobin due both to a fourfold higher rate of auto-oxidation and to a approximately 600-fold lower affinity for hemin. The role of His61(E7) in regulating oxygen binding was examined by site-directed mutagenesis. Replacement of His(E7) with Ala, Val or Leu causes little change in the equilibrium constant for O2 binding to soybean Lba, whereas the same mutations in sperm whale myoglobin cause 50 to 100-fold decreases in K(O2). These results show that, at neutral pH, hydrogen bonding with His(E7) is much less important in regulating O2 binding to the soybean protein. The His(E7) to Phe mutation does cause a significant decrease in K(O2) for Lba, apparently due to steric hindrance of the bound ligand. The rate constants for O2 dissociation from wild-type and native Lba decrease significantly with decreasing pH. In contrast, the O2 dissociation rate constants for mutants with apolar E7 residues are independent of pH, suggesting that hydrogen bonding to the distal histidine residue in the native protein is enhanced under acid conditions. All of these results support the hypothesis that the high affinity of Lba for oxygen and other ligands is determined primarily by enhanced accessibility and reactivity of the heme group.     

Pregnancy outcomes following false-positive multiple marker screening tests

Pregnancy outcomes in women with a false-positive midtrimester multiple marker screening test (MMST) were reviewed. A genetic database was used to identify all women > or = age 30 who had a MMST at 15-20 weeks of gestation, a targeted ultrasound, and amniocentesis, and complete pregnancy outcome data. All patients with an abnormal fetal ultrasound (US) or karyotype were excluded. The incidence of adverse outcomes (defined as fetal death, preterm delivery, or a birth weight less than the 10th percentile for gestational age), in those women with a positive MMST (risk of Down's syndrome > or = 1:190) was compared to the incidence of adverse outcomes in control women with negative MMST. Chi-square analysis and Fisher's exact tests were used for comparisons as appropriate. Complete data was available from 1135 women. Seventy-seven percent were over age 35. Two hundred and forty-six women (22%) had a positive multiple marker test. No significant differences in outcomes were discovered after comparisons to controls: fetal death 1 of 246 (0.4%) versus 12 of 889 (1.3%), p = 0.32; preterm delivery 32 of 246 (13.0%) versus 147 of 889 (16.5%), p = 0.17; birth weight less than the 10th percentile, 9 of 246 (3.7%) versus 30 of 889 (3.4%), p = 0.83. Our data suggest that women > or = age 30 with a false-positive MMST and a normal midtrimester obstetrical sonogram are not at an increased risk for adverse pregnancy outcomes in later gestation.