The Higher Structure of Chromatin in the LCR of the β-Globin Locus Changes during Development

The β-globin locus control region (LCR) is able to enhance the expression of all globin genes throughout the course of development. However, the chromatin structure of the LCR at the different developmental stages is not well defined. We report DNase I and micrococcal nuclease hypersensitivity, chromatin immunoprecipitation analyses for histones H2A, H2B, H3, and H4, and 3C (chromatin conformation capture) assays of the normal and mutant β-globin loci, which demonstrate that nucleosomes at the DNase I hypersensitive sites of the LCR could be either depleted or retained depending on the stages of development. Furthermore, MNase sensitivity and 3C assays suggest that the LCR chromatin is more open in embryonic erythroblasts than in definitive erythroblasts at the primary- and secondary-structure levels; however, the LCR chromatin is packaged more tightly in embryonic erythroblasts than in definitive erythroblasts at the tertiary chromatin level. Our study provides the first evidence that the occupancy of nucleosomes at a DNase I hypersensitive site is a developmental stage-related event and that embryonic and adult cells possess distinct chromatin structures of the LCR.     

Cloning of the cDNA and Genes for the Hamster and Human β2-Adrenergic Receptors

Abstract

The adenylate cyclase-stimulatory β₂-adrenergic receptor has been purified to apparent homogeneity from hamster lung. Partial amino acid sequence obtained from isolated CNBr peptides was used to clone the gene and cDNA for this receptor. The predicted amino acid sequence for the hamster β₂-adrenergic receptor revealed that the protein consists of a single polypeptide chain of 418 aa with consensus N-glycosylation and phosphorylation sites predicted by previous in vitro data. The most striking feature of the receptor protein however, is that it contains seven stretches of hydrophobic residues similar to the proposed seven transmembrane segments of the light receptor rhodopsin. Significant amino acid homology (30-35%) can be found between the hamster β₂-adrenergic receptor and rhodopsin within these putative membrane spanning regions. Using a hamster β₂-adrenergic receptor probe, the gene and cDNA for the human β₂-adrenergic receptor were isolated, revealing a high degree of homology (87%) between the two proteins from different species. Unlike the genes encoding the family of opsin pigments, of which rhodopsin is a member, the genes encoding both hamster and human β₂-adrenergic receptors are devoid of introns in their coding as well as 5′ and 3′ untranslated nucleotide sequences. The cloning of the genes and the elucidation of the aa sequences for these G-protein coupled receptors should help to determine the structure-function as well as the evolutionary relationship of these proteins.     

Structure and dynamics of oligomeric intermediates in β2-microglobulin self-assembly

β₂-Microglobulin is a 99-residue protein with a propensity to form amyloid-like fibrils in vitro which exhibit distinct morphologies dependent on the solution conditions employed. Here we have used ion mobility spectrometry-mass spectrometry to characterize the oligomeric species detected during the formation of worm-like fibrils of β₂-microglobulin at pH 3.6. Immediately upon sample dissolution, β₂-microglobulin monomer and oligomers—the latter ranging in size from dimer to hexamer—are present as a pool of rapidly interconverting species. Increasing the ionic strength of the solution initiates fibril formation without a lag-phase whereupon these oligomers become more stable and higher-order species (7-mer to >14-mer) are observed. The oligomers detected have collision cross-sectional areas consistent with a linearly stacked assembly comprising subunits of native-like volume. The results provide insights into the identity and properties of the transient, oligomeric intermediates formed during assembly of worm-like fibrils and identify species that differ significantly from the oligomers previously characterized during the nucleated assembly of long, straight fibrils. The data presented demonstrate the interrelationship between different fibril-forming pathways and identify their points of divergence.     

Implication of C-Terminal Deletion on the Structure and Stability of Bovine β-casein

Bovine β-casein (β-CN) with its C-terminal truncated by chymosin digestion, β-CN-(f1-192), was examined and characterized using circular dichroism (CD) under various temperature conditions. CONTIN/LL analysis of the CD data revealed significant secondary structure disruption in β-CN-(f1-192) relative to its parent protein,β-CN, in the temperature range (5° to 70°C) studied. Near-UV CD spectra indicated significant temperature dependent structural changes. Analytical ultracentrifugation results showed significant reduction but not complete abolishment of self-association in β-CN-(f1-192) compared to whole β-casein at 2°–37°C. Furthermore, binding experiments with the common hydrophobic probe – 8-anilino-1- naphthalene sulfonic acid (ANS) illustrated that β-CN-(f1-192) is nearly incapable of binding to ANS relative to whole β-CN, suggesting a nearly complete open overall tertiary structure brought about by the C-terminal truncation. It has been demonstrated clearly that the tail peptide β-CN-(f193-209) is important in maintaining the hydrophobic core of β-CN but the residual association observed argues for a minor role for other sites as well.     

Genetic Variability of the β-Tubulin Genes in Benzimidazole-Susceptible and -Resistant Strains of Haemonchus Contortus

Benzimidazole anthelmintics are the most common chemotherapeutic agents used to remove intestinal helminths from farm animals. The development of drug resistance within helminth populations is wide-spread and can render these drugs essentially useless. The mechanism of benzimidazole resistance appears to be common to many species ranging from fungi to nematodes and involves alterations in the genes encoding β-tubulin. During the selection process resulting in resistance, there must be quantitative changes in the population gene pool. Knowledge of these changes would indicate the mechanisms underlying the spread of resistance in the population, which in turn could be used to design more effective drug administration strategies. To this end we have identified allelic variation at two β-tubulin genes in Haemonchus contortus using restriction map analysis of individual adults. Extremely high levels of variation were identified at both loci within a susceptible strain. In two independently derived benzimidazole resistant strains, allele frequencies at both loci were significantly different from the susceptible strain but not from each other. The same alleles at both loci, in both resistant strains, were favored by selection with benzimidazoles, suggesting that both loci are involved in determining benzimidazole resistance. These data confirm that changes in allele frequency, rather than novel genetic rearrangements induced by exposure to the drug, explain the changes associated with benzimidazole resistance. These results also show that any DNA based test for the development of benzimidazole resistance must take into account the frequency of alleles present in the population and not simply test for the presence or absence of specific allelic types.     

Investigations on the genetics and population genetics of the β2 polymorphism

Summary The results of studies on 49 families with 107 children and various populations of Caucasoid, Negroid and Mongoloid origin concerning the genetics and population genetics of the ₂-glycoprotein I polymorphism are reported. In general the genetical model proposed by Cleve (1968) is confirmed: two autosomal alleles Bg^N and Bg^D controlling the phenotypes Bg N-N, Bg N-D and Bg D-D. However, divergences from this model were found in two families. They indicate the assumption of non-genetic factors influencing the phenotype expression rather than more complicated genetical control mechanisms. Within Caucasoid populations phenotype and gene frequencies show almost a homogeneous distribution. This racial stock is striking due to a significant higher ₂-glycoprotein I concentration in serum as compared to Negroids and Mongoloids. In connection with this, these racial stocks differ obviously in the gene frequencies: Caucasoids Bg^N=0.937, Negroids=0.742, Mongoloids =0.780; resp. Bg^D=0.063, 0.258, 0.220.Supported by the Deutsche Forschungsgemeinschaft.D 77.     

Chronic effects of β2 agonists on body composition and protein synthesis in the rat

Chronic treatment of rats with the ₂-adrenergic agonists clenbuterol and fenoterol over 16–19 d raised energy intake, expenditure, and body weight gain but did not affect fat or energy deposition, and body protein gain was increased by 50 and 18%, respectively. Both drugs increased the protein content and mitochondrial GDP-binding capacity of brown adipose tissue. Clenbuterol did not affect plasma insulin, growth hormone, or triiodothyronine levels, although insulin levels were reduced by fenoterol. Both drugs caused hypertrophy of skeletal muscle (gastrocnemius), and muscle protein synthesis in vivo (fractional rate) was elevated by 34 and 26% in clenbuterol and fenoteroltreated rats, respectively.     

SMN, Gemin2 and Gemin3 Associate with β-Actin mRNA in the Cytoplasm of Neuronal Cells In Vitro

Childhood spinal muscular atrophy is caused by a reduced expression of the survival motor neuron (SMN) protein. SMN has been implicated in the axonal transport of β-actin mRNA in both primary and transformed neuronal cell lines, and loss of this function could account, at least in part, for spinal muscular atrophy onset and pathological specificity. Here we have utilised a targeted screen to identify mRNA associated with SMN, Gemin2 and Gemin3 in the cytoplasm of a human neuroblastoma cell line, SHSY5Y. Importantly, we have provided the first direct evidence that β-actin mRNA is present in SMN cytoplasmic complexes in SHSY5Y cells.     

Current and Novel Aspects on the Non-lysosomal β-Glucosylceramidase GBA2

The non-lysosomal β-glucosylceramidase GBA2 (EC3.2.1.45, GH116) is ubiquitously expressed in various mammal tissues and cell types where it catalyzes the hydrolysis of glucosylceramide into glucose and ceramide. Although it has been known for many years that the central nervous system is the main site of GBA2 expression and activity, little information has been available so far on the role of this protein in the neuronal development, senescence and homeostasis. In the present review, we summarize the state-of-the art of this enzyme and in particular, we focus on the current knowledge on its structure, its physico-chemical properties and its subcellular localization. Data on the involvement of GBA2 in physiopathological processes are also described, with particular emphasis on the studies that have indicated the direct involvement of GBA2 in neuronal development and neurological disorders. A discussion of some open questions and future perspectives related to GBA2 are finally reported. We conclude that further investigations on this β-glucosylceramidase will provide new clues about the physiology of the central nervous system.     

Preliminary studies on the characteristics of phenylalanine and β-methyl-glucoside transport in the tench intestine in vitro

• 1.1. The unidirectional transepithelial fluxes of L-phenylalanine, β-methyl-D-glucoside and sodium ions across emusculated sheets of tench mid-intestine were determined in flux chambers.
• 2.2. No net sodium flux was detectable, but phenylalanine was preferentially transferred from the mucosal to the serosal fluid.
• 3.3. There was also a net movement of β-methyl-glucoside towards the serosal medium, but it was much smaller than that of phenylalanine.
• 4.4. This transport was accompanied by an accumulation of each substrate from the mucosal medium into the tissue to a similar level and against a concentration gradient.
• 5.5. The poor transfer of the monosaccharide into the serosal medium could therefore be attributed to a low permeability of the baso-lateral membrane of the enterocyte for this substance.
• 6.6. The influx of L-phenylalanine and of β-methyl-d-glucoside into the epithelial cells of tench midintestine was examined by incubating slices of emusculated intestine in radioactively-labelled solutions of the substrate for 2 min.
• 7.7. The steady-state uptake was assessed after similar incubations lasting 45 min.
• 8.8. Phenylalanine influx obeys the Michaelis-Menten equation with a K_m of 2.9 mM and is dependent on the presence of sodium ions in the incubation medium.
• 9.9. β-Methyl-glucoside influx reveals the same characteristics with a K_m of 2.0 mM but a considerably lower V_max; in addition, it is inhibited by galactose.
• 10.10. The influx of both substrates is reduced by harmaline, which also inhibits the uptake of radioactive sodium by this preparation.
• 11.11. The steady-state uptake of β-methyl-glucoside is also inhibited by ouabain and by 2.4-dinitrophenol.
• 12.12. These results suggest that the mechanisms for sodium-dependent influx of monosaccharides and neutral amino-acids in the tench intestine are similar to those found in mammalian tissues.
• 13.13. The principal difference appears to involve the release of monosaccharides across the baso-lateral membrane of the enterocyte.

     

Effect of Zn（Ⅱ） on the Structure and Biological Activity of Natural β-NGF

Only β-NGF, the subunit of the 7S NGF complex, exhibits NGF activity, but the function ofthe zinc ion in native β-NGF has received little attention. Flameless atomic absorption spectroscopy (FAAS)measurements reveal that native β-NGF contains Zn(Ⅱ) with a Zn(Ⅱ)/β-NGF stoichiometry of 1 : 14.6.The presence of Zn(Ⅱ) in the native molecule results in significant changes of the secondary structure andlocal tertiary structure around Trp(s) with respect to those of apo β-NGF, as suggested by spectra offluorescence and circular dichrosim. Stopped-flow studies show that there are at least two steps during theinteraction of Zn(Ⅱ) with the apo form. In comparison with its apo form, the native β-NGF shows a higherability to trigger the proliferation of TF1 cells and mediate the survival of PC 12. Thus it is most likely that thestructural changes caused by the presence of Zn(II) directly lead to the increase in the biological activity of β-NGE All results indicate that Zn(Ⅱ) in native β-NGF plays an important role in the structure and thebiological activity of the protein.     

Structure of soybean β-cyanoalanine synthase and the molecular basis for cyanide detoxification in plants

Plants produce cyanide (CN-) during ethylene biosynthesis in the mitochondria and require β-cyanoalanine synthase (CAS) for CN- detoxification. Recent studies show that CAS is a member of the β-substituted alanine synthase (BSAS) family, which also includes the Cys biosynthesis enzyme O-acetylserine sulfhydrylase (OASS), but how the BSAS evolved distinct metabolic functions is not understood. Here we show that soybean (Glycine max) CAS and OASS form α-aminoacrylate reaction intermediates from Cys and O-acetylserine, respectively. To understand the molecular evolution of CAS and OASS in the BSAS enzyme family, the crystal structures of Gm-CAS and the Gm-CAS K95A mutant with a linked pyridoxal phosphate (PLP)-Cys molecule in the active site were determined. These structures establish a common fold for the plant BSAS family and reveal a substrate-induced conformational change that encloses the active site for catalysis. Comparison of CAS and OASS identified residues that covary in the PLP binding site. The Gm-OASS T81M, S181M, and T185S mutants altered the ratio of OASS:CAS activity but did not convert substrate preference to that of a CAS. Generation of a triple mutant Gm-OASS successfully switched reaction chemistry to that of a CAS. This study provides new molecular insight into the evolution of diverse enzyme functions across the BSAS family in plants.     

The Effect of Dietary β-Sitosterol and β-Sitostanol on the Metabolism of Cholesterol in Rats

Effects of dietary β-sitosterol (S) and β-sitostanol (HS) on the metabolism and fate of labeled cholesterol intravenously injected were compared in rats fed diets high in cholesterol. Kinetic behavior of the decay curve for serum cholesterol in the HS supplemented (C + HS) group approximated to that in the cholesterol-free (control) group. The largest dilution of the label was observed in rats of the cholesterol (C) group and the least in the C + HS group, the C + S group being intermediate. The specific activity of hepatic cholesterol was in the decreasing order of the C + HS, C + S and C groups, while the situation was reversed when expressed in terms of net incorporation. Thus, cholesterol pool seemed to be much smaller in the C + HS group than in the C + S group.

In a long term feeding experiment with diets free of cholesterol, HS exhibited significantly greater hypocholesterolemic activity than S did.

These data, together with those reported previously, indicated that inhibitory effect on the absorption of both endogenous and exogenous cholesterol was much more greater in HS than in S.     

Positions of β2 and β3 subunits in the large-conductance calcium- and voltage-activated BK potassium channel

Large-conductance voltage- and Ca²⁺-gated K⁺ channels are negative-feedback regulators of excitability in many cell types. They are complexes of α subunits and of one of four types of modulatory β subunits. These have intracellular N- and C-terminal tails and two transmembrane (TM) helices, TM1 and TM2, connected by an ∼100-residue extracellular loop. Based on endogenous disulfide formation between engineered cysteines (Cys), we found that in β2 and β3, as in β1 and β4, TM1 is closest to αS1 and αS2 and TM2 is closest to αS0. Mouse β3 (mβ3) has seven Cys in its loop, one of which is free, and this Cys readily forms disulfides with Cys substituted in the extracellular flanks of each of αS0–αS6. We identified by elimination mβ3-loop Cys152 as the only free Cys. We inferred the disulfide-bonding pattern of the other six Cys. Using directed proteolysis and fragment sizing, we determined this pattern first among the four loop Cys in β1. These are conserved in β2–β4, which have four additional Cys (eight in total), except that mβ3 has one fewer. In β1, disulfides form between Cys at aligned positions 1 and 8 and between Cys at aligned positions 5 and 6. In mβ3, the free Cys is at position 7; position 2 lacks a Cys present in all other β2–β4; and the disulfide pattern is 1–8, 3–4, and 5–6. Presumably, Cys 2 cross-links to Cys 7 in all other β2–β4. Cross-linking of mβ3 Cys152 to Cys substituted in the flanks of αS0–S5 attenuated the protection against iberiotoxin (IbTX); cross-linking of Cys152 to K296C in the αS6 flank and close to the pore enhanced protection against IbTX. In no case was N-type inactivation by the N-terminal tail of mβ3 perturbed. Although the mβ3 loop can move, its position with Cys152 near αK296, in which it blocks IbTX binding, is likely favored.