Cooperative thermal transitions of bovine and human apo-alpha-lactalbumins: evidence for a new intermediate state

The thermal denaturation of bovine and human apo-alpha-lactalbumins at neutral pH has been studied by intrinsic protein fluorescence, circular dichroism (CD), and differential scanning microcalorimetry (DSC) methods. Apo-alpha-lactalbumin possesses a thermal transition with a midpoint about 25-30 degrees C under these conditions (pH 8.1, 10 mM borate, 1 mM EGTA), which is reflected in changes in both fluorescence emission maximum and quantum yield. However, the CD showed a decrease in ellipticity at 270 nm with a midpoint at about 10-15 degrees C, while DSC shows the transition within the region of 15-20 degrees C. The non-coincidence of transition monitored by different methods suggests the existence of an intermediate state in the course of the thermal denaturation process. This intermediate state is not the classical molten globule state which occurs at higher temperature (i.e. denatured state at these conditions) [D.A. Dolgikh, R.I. Gilmanshin, E.V. Brazhnikov, V.E. Bychkova, G.V. Semisotnov, S.Y. Venyaminov and O.B. Ptitsyn, FEBS Letters, 136 (1981) 311-315] and has physical properties intermediate between the native and molten globule states.     

Molecular modeling of immunoglobulin light chains implicates hydrophobic residues in non-amyloid light chain deposition disease

Light chain deposition disease is a severe complication of certain immunoproliferative disorders, due to the secretion of a monoclonal light chain which precipitates close to basement membranes of several tissues. A kappa isotype restriction and an unusual frequency of a variable region subgroup (VkappaIV) suggest that precise structural features govern the propensity of pathogenic light chains to precipitate in extracellular spaces. We studied primary structures of light chains from six patients with light chain deposition disease in comparison with light chains from other pathological conditions. Sequence alignment revealed the presence of certain amino acids only in light chain deposition disease, in particular non-polar replacing hydrophilic residues. To determine the role of these residues, structures of the variable domain from four kappa chains belonging to VkappaI and VkappaIV subgroups responsible for deposition disease were modeled using known immunoglobulins as templates. The most evident structural features shared by all pathogenic light chains were hydrophobic residues exposed to the solvent in complementarity determining regions 1 or 3. In contrast to immunoglobulin light chain- related amyloidosis, where deposition of organized material might be due to electrostatic interactions between light chain dimers, hydrophobic interactions could enhance amorphous precipitation in non- amyloid light chain deposition disease.      

Carotenoid composition and vitamin A value of Brazilian loquat (Eriobotrya japonica Lindl.)

Six sample lots of loquat commercialized in Campinas, Brazil were analyzed for their carotenoid composition beta-carotene (7.8 micrograms/g), zeta-carotene (0.1 micrograms/g), neurosporene (1.1 micrograms/g), beta-cryptoxanthin (4.8 micrograms/g), 5,6-monoepoxy-beta-cryptoxanthin (0.6 micrograms/g), violaxanthin (1.6 micrograms/g), (0.8 micrograms/g) and auroxanthin (0.9 micrograms/g) were identified beta-Carotene and beta-cryptoxanthin were the principal pigments, being responsible for 44% and 27%, respectively, of the total carotenoid content (17.6 micrograms/g). Both were also the principal contributors to the vitamin A value of 175 RE/100g. The carotenoid composition of the Brazilian loquat resembles that of the Japanese loquat variety Tanaka.     

Isolated left superior vena cava to the left atrium with situs solitus and dextrocardia: extracardiac repair facilitated by juxtaposition of the atrial appendages

We describe an extremely unusual case, with isolated left superior vena cava to the left atrium, normal intracardiac anatomy, and left juxtaposition of the atrial appendages. Surgical repair was performed without cardiopulmonary bypass by anastomosing the left superior cava to the superior aspect of the right atrial appendage, and was facilitated by the ectopic location of the appendage.     

MMTV-Fgf8 transgenic mice develop mammary and salivary gland neoplasia and ovarian stromal hyperplasia

Prior studies have identified Fibroblast Growth Factor-8 (Fgf8) as a possible proto-oncogene in mouse mammary tumorigenesis. We now report on the generation of two types of Fgf8 transgenic mice that each utilize the mouse mammary tumor virus (MMTV) promoter. The first transgene (MMTV-Fgf8b) results in the overexpression of the FGF8b isoform exclusively. Male and female MMTV-Fgf8b transgenic mice are viable and fertile. RNA for FGF8b is detected in mammary gland and salivary gland tissues of transgenic mice by Northern blot analysis. Nearly 85% of breeding transgenic female mice developed mammary lobular adenocarcinomas by 12 months of age, while no tumors developed in non-transgenic littermates. Salivary gland tumors occurred in some animals, always in association with mammary tumors. Several MMTV-Fgf8b transgenic mice had lung metastases at necropsy. The second transgene (MMTV-Fgf8) uses the entire Fgf8 gene and potentially encodes all FGF8 isoforms. Fgf8 is expressed by this transgene in several tissues in addition to those described above, notably the ovaries. The two MMTV-Fgf8 founders developed mammary ductal adenocarcinomas at five and eight months of age, and both displayed ovarian stromal hyperplasia. The founders expressing either transgene did not successfully nurse their pups. These results demonstrate that production of FGF8b, and possibly other FGF8 isoforms, in the mammary and salivary glands contributes to oncogenesis, and that ovarian expression results in stromal hyperplasia.     

Bacteriophage T7 DNA ligase. Overexpression, purification, crystallization, and characterization

The bacteriophage T7 DNA ligase gene was amplified using polymerase chain reaction-based methods and cloned into a T7 promoter-based expression vector. The protein was overexpressed to greater than 15% of total soluble protein and purified to homogeneity, yielding 60-70 mg of protein per liter of bacterial culture. An initial physical and biochemical characterization of the enzyme reveals that it exists as a monomer and can ligate nicked, cohesive, and blunt-ended DNA fragments. Inhibition of the enzyme activity by a nonhydrolyzable ATP analogue was also investigated. The enzyme has been crystallized from methoxypolyethylene glycol. The crystals are of the orthorhombic space group P2(1)2(1)2 and diffract to 2.6 A. The unit cell dimensions are a = 66.1 A, b = 87.6 A, and c = 78.6 A, with one monomer in the asymmetric unit (Vm = 2.77 A3/Da). This is the first member of the DNA ligase family of enzymes to be crystallized.