Proteolytic specificity of chicken cathepsin L on bovineβ-casein

The proteolytic specificity of chicken cathepsin L was studied using bovine -casein as substrate. The peptide mixtures obtained after various times of hydrolysis were separated by RP-HPLC and ten peptides were identified. Chicken cathepsin L accepts proline residues in all positions except P ₁ . Looking at the amino acid residues on the amino side of the scissile bond we found three times the Tyr-Pro pair at P ₁ –P ₂ positions and that the S ₁ subsite can interact with modified amino acids such as phosphoserine.Abbreviations RP-HPLC reverse phase high performance liquid chromatography - NMec N-methyl coumarylamide - TEA triethylamine - TFA trifluoroacetic acid     

Regulation of Brain and Cerebrospinal Fluid Calcium by Brain Barrier Membranes Following Vitamin D-Related Chronic Hypo- and Hypercalcemia in Rats

Male Fischer-344 rats, 21 days old, were fed diets containing 0 (LOD), 2,200 (CONT), or 440,000 (HID) international units of vitamin D3 per kilogram for 12 weeks. [Ca] was measured in plasma, CSF, brain, and choroid plexus. In addition, 45Ca and 36Cl transfer coefficients (KCa and KCl) for uptake from blood into CSF and brain were determined. Although plasma ionized [Ca]s in LOD and HID rats were 50% and 136%, respectively, of values in CONT animals, CSF and brain [Ca]s ranged from only 85% to 110% of respective CONT values. Choroid plexus [Ca] was increased by 37% after HID diet, but was decreased only 10% after LOD. KCa values at CSF, parietal cortex, and pons-medulla were negatively correlated with plasma ionized [Ca], whereas KCl values at CSF and brain were not different between the diet groups. The findings demonstrate that central nervous system [Ca] is maintained during chronic hypo- or hypercalcemia by saturable transport of Ca at brain barrier membranes. This transport does not seem to involve modulation by 1,25-dihydroxyvitamin D3.     

Strand targeting signal(s) for in vivo mutation avoidance by post-replication mismatch repair in Escherichia coli

Summary The involvement of GATC sites in directing mismatch correction for the elimination of replication errors in Escherichia coli was investigated in vivo by analyzing mutation rates for a gene carried on a series of related plasmids that contain 2, 1 and 0 such sites. This gene encoding chloramphenicol acetyl transferase (Cat protein) was inactivated by a point mutation. In vivo mutations restoring resistance to chloramphenicol were scored in mismatch repair proficient (mut ⁺) and deficient (mutHLS^-) strains. In mut ⁺ cells, reduction of GATC sites from 2 to 0 increased mutation rates approximately 10-fold. Removal of the GATC site distal to the cat ^- mutation increased the rate of mutation less than 2-fold, indicating that mismatch repair can proceed normally with a single site. The mutation rate increased 3-fold after removal of the GATC site proximal to the mutation. In the absence of a GATC site, mutL^- and mutS^- strains exhibited a 2- to 3-fold increased mutation rate as compared to isogenic mutH^- and mut ⁺ strains. This indicates that 50%–70% of replication errors can be corrected in a mutLS-dependent way in the absence of any GATC site to target mismatch correction to newly synthesized DNA strands. Other strand targeting signals, possibly single strand discontinuities, might be used in mutLS-dependent repair     

Formation of wheat protein bodies: Involvement of the Golgi apparatus in gliadin transport

Developing wheat (Triticum aestivum L.) endosperm was examined using ultrathin sections prepared from tissues harvested at 5, 9, 16 and 25 d after flowering. Protein bodies were evident by 9 d and displayed a variety of membranous structures and inclusions. The Golgi apparatus was a prominent organelle at all stages, and by 9 d was associated with small electron-dense inclusions. By immunocytochemical techniques, gliadin (wheat prolamine) was localized within these vesicles and in homogeneous regions of protein bodies, but not in the lumen of the rough endoplasmic reticulum. The protein bodies appear to enlarge by fusion of smaller protein bodies resulting in larger, irregular-shaped organelles. The affinity of the Golgi-derived vesicles for gliadin-specific probes during the period of maximal storage-protein synthesis and deposition indicates that this organelle includes the bulk, if not all, of the gliadin produced. The involvement of the Golgi apparatus in the packaging of gliadins into protein bodies indicates a pathway which differs from the mode of prolamine deposition in other cereals such as maize, rice and sorghum, and resembles the mechanism employed for the storage of rice glutelin and legume globulins.Abbreviations ER endoplasmic reticulum - IgG immunoglobulin G - DAF days after flowering     

Cell surface expression of 4β-galactosyltransferase accompanies rat parotid gland acinar cell transition to growth

Rat parotid gland acinar cells stimulated to divide by a chronic regimen of isoproterenol demonstrate a dramatic increase in the synthesis of the glycosyltransferase 4β-galactosyltransferase. A plasma membrane localization for much of the increase in 4β-galactosyltransferase was determined by density gradient membrane fractionation. Golgi-enriched fractions showed no increase in specific activity, while plasma membrane activity increased 40-fold. This selective increase at the cell surface was confirmed by immunofluorescence of intact, nonpermeabilized cells from treated glands, using a monospecific antibody prepared against the purified bovine milk transferase. In detergent-permeabilized cells staining of nontreated cells was seen only as groups of perinuclear vesicles, presumed to be Golgi apparatus. In isoproterenol-treated and permcabilized cells both presumptive Golgi and cell surface staining was apparent. Enzyme assays performed on intact cells established that the enzyme's active site was oriented to the exterior of the cells. The transferase could be detected as early as 3 hr after the primary challenge with isoproterenol. Pretrcatment of rats with cycloheximide prevented its appearance.     

Zinc fingers and other domains cooperate in binding of Drosophila sry beta and delta proteins at specific chromosomal sites.

The closely related Drosophila serendipity (sry) beta and delta zinc finger proteins display consensus in vitro DNA recognition sequences differing by 4 of 13 nucleotide positions and bind in vivo to distinct sets of sites on polytene chromosomes. We compared the pattern of in vivo chromosomal binding of deleted forms of the sry delta protein fused to beta-galactosidase and expressed in Drosophila transgenic lines. Results show that the carboxy-terminal DNA-binding finger domain is required and sufficient for binding at specific chromosomal sites but that this binding does not nearly reproduce the wild-type pattern. An NH2-terminal domain of the sry delta protein is essential to its specificity of in vivo interaction with chromatin. In vitro and in vivo experiments using reciprocal finger swap between the sry beta and delta proteins suggest that the in vivo specificity is dependent on selective protein-protein contacts at defined chromosomal sites, in addition to DNA specific recognition.     

Solubility and reactivity of caseins and beta-lactoglobulin in protic solvents.

The study of the solubility of unstructured proteins (_S1-, -, and -casein) and well-structured globulin (-lactoglobulin) in low water binary solvent systems demonstrated the crucial importance of solvent polarity and neutralization of protein polar functions on the final outcome of solubility experiments. The solubilities up to 38, 56, and 96% in CHCl₃/CH₃OH (1/1, v/v) acidified with HCl and up to 5, 10, and 25% in CHCl₃/CH₃OH (1/1, v/v) in the presence of triethylamine (TEA) were obtained for -, _S1-, and -casein, respectively. The importance of protein charge neutralization was apparent when the solubilization was performed in basified CHCl₃/CH₃OH media, giving the optimal results when the studied proteins were brought before to their isoionic point. The maximum solubility of -casein at its pI in 30–70% methanol in CHCl₃ was reaching 50–60% with triethylamine (TEA) added. -lactoglobulin could be solubilized up to 70% in CHCl₃/CH₃OH (7/3, v/v) acidified with HCl and up to 40% in CHCl₃/CH₃OH (3/7, v/v) in the presence of TEA. The observed yield of reductive alkylation of -lactoglobulin was much higher (98%) when performed in studied solvent system than in aqueous conditions (75%). Apparently, steric hindrance of the well-folded -barrel (in aqueous conditions) structure masks the portion of -NH₂ groups. In the case of unstructured aqueous media -casein, 90% alkylation yields were obained in organic and aqueous conditions.     

Absence of Turner stigmata in a 46,XYp-female

A 46,XY female patient with streak gonads and a large deletion of Yp is described. The deletion included the Y chromosomal genes SRY, ZFY, and RPS4Y. The patient did not display any Turner stigmata, such as webbing of the neck, cardiac or other abnormalities. The findings argue against an important role of RPS4Y in the prevention of Turner stigmata in males and are consistent with a role of SRY in testis differentiation in humans.     

The Machado-Joseph disease locus is different from the spinocerebellar ataxia locus (SCA1).

Machado-Joseph disease (MJD) is an autosomal dominant neurodegenerative spinocerebellar ataxia that has been described primarily in families of Azorean or Portuguese descent. MJD and chromosome 6p-linked spinocerebellar ataxia (SCA1) are difficult to differentiate clinically, and it has been suggested that they may be allelic variants of the same disorder. We have tested MJD families for linkage to six DNA sequence polymorphisms located on chromosome 6p, including the highly informative dinucleotide repeat, D6S89. Seventeen centimorgans telomeric to and 41 cM centromeric to D6S89, a region that includes the SCA1 locus reported to be within 3 cM of D6S89, have been excluded. These data provide conclusive evidence that MJD and SCA1 are nonallelic.     

Cyclic behavior of plum curculio,Conotrachelus nenuphar (Herbst) (Coleoptera: Curculionidae), within caged dwarf apple trees in spring

From 12 to 19 May 1987, during Morspur apple bloom, 21 radioactively labeled (⁶⁵ Zn) adult plum curculios, Conotrachelus nenuphar (Herbst), were released within a field cage containing four dwarf apple trees and located three times a day. A technique was developed for quickly obtaining (x, y, z)coordinates of location for adults foraging within apple trees. Cyclic patterns of behavior were detected using spectral analysis procedures. Over 70% of plum curculios exhibited diel periodicity with respect to activity and rate of movement, 36% exhibited such periodicity with respect to presence in the trees, and 27% with respect to movements from the center to the periphery of the canopy. Presence in fruit clusters, height in the trees, and movements along east-west and north-south axes showed little or no periodicity. Factors triggering cyclic behavior and practical implications of the results are discussed.