Theory of cooperative transitions in protein molecules. I. Why denaturation of globular protein is a first-order phase transition

A theory of equilibrium denaturation of proteins is suggested. According to this theory, a cornerstone of protein denaturation is disruption of tight packing of side chains in protein core. Investigation of this disruption is the object of this paper. It is shown that this disruption is an "all-or-none" transition (independent of how compact is the denatured state of a protein and independent of the protein-solvent interactions) because expansion of a globule must exceed some threshold to release rotational isomerization of side chains. Smaller expansion cannot produce entropy compensation of nonbonded energy loss; this is the origin of a free-energy barrier (transition state) between the native and denatured states. The density of the transition state is so high that the solvent cannot penetrate into protein in this state. The results obtained in this paper make it possible to present in the following paper a general phase diagram of protein molecule in solution.     

1-Methyladenine in urine of an adenosine deaminase-deficient adult without immunodeficiency

Procedures are described for the isolation and identification of 1-methyladenine from the urine of an adult female with adenosine deaminase deficiency but no immunodeficiency. Evidence is provided indicating that much of the usual urinary excretion product, 1-methyladenosine, is converted to 1-methyladenine in this subject prior to excretion. Since the nucleoside phosphorylases present in normal individuals do not act on 1-methyladenosine, this suggests that a phosphorylase with unusual properties is present in this adenosine deaminase-deficient subject. A possible role for this phosphorylase in removal of deoxyadenosine in this subject is discussed.     

Establishment of the primary imprint of the HYMAI/PLAGL1 imprint control region during oogenesis

Imprinting within domains occurs through epigenetic alterations to imprinting centers (ICs) that result in the establishment of parental-specific differences in gene expression. One candidate IC lies within the imprinted domain on human chromosome region 6q24. This domain contains two paternally expressed genes, the zinc finger protein gene PLAGL1 (ZAC/LOT1) and an untranslated mRNAcalled HYMAI. The putative IC overlaps exon 1 of HYMAI and is differentially methylated in somatic tissues. In humans, loss of methylation within this region is seen in some patients with transient neonatal diabetes mellitus, and hypermethylation of this region is found in ovarian cancer and is associated with changes in expression of PLAGL1, suggesting that it plays a key role in regulating gene expression. Differential methylation within this region is conserved in the homologous region on mouse chromosome 10A and is present on the maternal allele. In this paper, we report that DNA methylation is established during the growth phase of oogenesis and that this coincides with the establishment of monoallelic expression from this region lending further support to the hypothesis that this region functions as an IC.     

Components and mechanisms of thermal hyperpnea.

The pattern of breathing during a hyperthermia-induced hyperventilation varies across different species. Thermal tachypnea is a first phase panting response adopted during hyperthermia when tidal volume is minimized and the frequency of breathing is maximized. Blood-gas tensions and pH are maintained during this hyperventilation, and the associated heat loss helps the animal regulate its body temperature. A second pattern of breathing adopted in hyperthermia is thermal hyperpnea; this response is the focus of this review. This form of hyperventilation is evident after an increase in core temperature and it is apparent in humans. Increases of tidal volume as well as frequency of breathing are evident during this response that results in a respiratory alkalosis. The cause of thermal hyperpnea is not resolved; evidence of the potential mechanisms underlying this response support that modulators of the response act in either a multiplicative or additive manner with body temperatures. The details of the designs and methodologies of the studies supporting or refuting these two views are discussed. A physiological rationale for thermal hyperpnea is presented in which it is suggested this response serves a heat-loss role and contributes to selective brain cooling in hyperthermic humans. Ongoing research in this area is focused on resolving the mechanisms underlying thermal hyperpnea and its contribution to cranial thermoregulation. The direct application of this research is for the care of febrile and hyperthermic patients.     

The relationship between the stiffness and the mineral content of bone

The modulus of elasticity (E) of bone increases very rapidly with increase in mineral content, and in this is atypical of most composite materials. It is proposed that this apparent anomaly is caused by the end-to-end fusion of apatite crystals as the matrix becomes saturated with mineral. There is electron microscopic evidence that this occurs. Calculations using a fairly simple model show that this mechanism could be effective in life.     

Splicing and transport of eukaryotic mRNAs: a theoretical model

Several years have already elapsed since the first discovery of splicing in eukaryotic mRNAs. In this process sections of the precursor mRNAs are spliced out (these are named introns) and the two remaining excised sites, 5′ and 3′ are ligated to form the mature mRNA chains.Very little is known about the splicing and ligation mechanism or about its location inside the cell. It is known to take place in the nucleus, but it is unknown whether it occurs inside the nuclear matter or on the surface of its membrane. Since nearly all eukaryotic messengers undergo splicing, this is a central question.From the theoretical point of view this is an intriguing problem. A lot of data have recently accumulated which have a bearing on this question. Based on current knowledge, this paper proposes a model in which splicing is carried out on the surface of the nuclear membrane and in concert with transport across it. It is suggested that the enzymes that take part in this process are loosely associated with the membrane pore complex. Evidence and results which are relevant to this question are given and discussed.     

The copper toxicosis gene product Murr1 directly interacts with the Wilson disease protein

Copper toxicosis in Bedlington terriers is an autosomal recessive disorder characterized by excessive hepatic copper accumulation in association with a marked decrease in biliary copper excretion. Recent genetic data have revealed that MURR1, a single copy gene on dog chromosome 10q26, is mutated in this disorder. This gene encodes a 190-amino acid open reading frame of unknown function that is highly conserved in vertebrate species. The Wilson disease protein is a copper transporting ATPase shown to play a critical role in biliary copper excretion. Here we demonstrate that the Wilson disease protein directly interacts with the human homologue of Murr1 in vitro and in vivo and that this interaction is mediated via the copper binding, amino terminus of this ATPase. Importantly, this interaction is specific for this copper transporter, a finding consistent with the observation that impaired copper homeostasis in affected terriers is confined to the liver. Our findings reveal involvement of Murr1 in the defined pathway of hepatic biliary copper excretion, suggest a potential mechanism for Murr1 function in this process, and provide biochemical evidence in support of the proposed role of the MURR1 gene in hepatic copper toxicosis.     

Even if the fetus is not a person,abortion is immoral: The impairment argument

Much of the debate about the ethics of abortion has centered on whether the fetus is a person. In an attempt to sidestep this complex issue, I argue that, even if the fetus is not a person, abortion is immoral. To arrive at this conclusion, I argue that giving a fetus fetal alcohol syndrome is immoral, and that if this is so, then killing the fetus is immoral. Roughly, this is because killing the fetus impairs it more than giving it fetal alcohol syndrome. Since abortion (in most cases) amounts to killing the fetus, this means that abortion (in most cases) is immoral. I defend the premises of this argument against a plethora of objections, concluding that they either do not work, or commit their proponent to a controversial position.     

The control of sexual identity in the Drosophila germline

Whether to be male or female is a critical decision in development. Nowhere is this more important than in the germ cells, which must produce either the sperm or eggs necessary for the perpetuation of the species. How does a germ cell make this decision and how is it executed? One thing that is clear is that this process is very different in germ cells compared with other cells of the embryo. Here, we explore how sexual identity is established in the Drosophila germline, how this affects other aspects of germ cell development and what studies in Drosophila can teach us about mammalian germ cells.     

On the 'rate of aging' in heterogeneous populations

It is well known that the rate of aging is constant for populations described by the Gompertz law of mortality. However, this is true only when a population is homogeneous. In this note, we consider the multiplicative frailty model with the baseline distribution that follows the Gompertz law and study the impact of heterogeneity on the rate of aging in this population. We show that the rate of aging in this case is a function of age and that it increases in (calendar) time when the baseline mortality rate decreases.     

Asparagine 81, an invariant glycosylation site near apyrase conserved region 1, is essential for full enzymatic activity of ecto-nucleoside triphosphate diphosphohydrolase 3

N-linked glycosylation is important for the function, cellular localization, and oligomerization of membrane-bound ecto-nucleoside triphosphate diphosphohydrolases (eNTPDases). NTPDase3 is a prototypical cell membrane-associated eNTPDase, which is equally related and enzymatically intermediate to the other two cell surface membrane NTPDases (NTPDase1 and 2). The protein sequence of NTPDase3 contains seven putative N-glycosylation sites located in the ecto-domain. Only one of these putative glycosylation sites, asparagine 81 in NTPDase3, which is located near apyrase conserved region 1 (ACR1), is invariant in all the cell surface membrane eNTPDases. Using site-directed mutagenesis, mutants were constructed to eliminate this highly conserved N-glycosylation site in NTPDase3. The results indicate that glycosylation at this position is essential for full enzymatic activity, with mutant ATPase activity decreased more than ADPase activity. Enzymatic deglycosylation of this site is shown to be responsible for the inactivation of the wild-type enzyme by treatment with peptide N-glycosidase-F. In addition, glycosylation of this conserved site is necessary for the stabilization/stimulation of nucleotidase activity upon treatment with the lectin concanavalin A. However, lack of glycosylation at this site did not result in large changes in tertiary or quaternary structure, as measured by Cibacron blue binding, chemical cross-linking, and native gel electrophoretic analysis. Since this N-glycosylation site is invariant in cell membrane eNTPDases, it is postulated that glycosylation of this residue near ACR1 is crucial for full enzymatic activity of the cell membrane NTPDases.     

INTRACELLULAR DISTRIBUTION OF ALKALINE PHOSPHATASE IN RAT LIVER CELLS

1. Cytochemical studies of the intracellular distribution of alkaline phosphatase in rat liver have been made, using a fractionation procedure recently developed in this laboratory (8) and a similar but modified method not described previously. Aqueous media were used in both cases. 2. The alkaline phosphatase was found to consist of two forms, one of which is strongly activated by magnesium and one of which is not sensitive to this metal. 3. The form of the enzyme that is not activated by magnesium occurs mainly in the nuclear fraction, where it seems to be rather firmly bound. Some of this form of the enzyme is also found in the microsomes, but very little if any occurs in the soluble supernatant fraction. 4. The form of alkaline phosphatase which is activated by magnesium occurs mainly in the soluble supernatant fraction, but what is believed are significant amounts also occur in nuclei. A significant portion of this form of the enzyme can be extracted from the isolated nuclei with cold, isotonic saline solution. Some activity of this form of the enzyme is also found in the microsomal fraction. 5. Mitochondria appear to contain relatively little alkaline phosphatase of either kind. 6. The concept of a porous nuclear membrane has been invoked to explain some of the results obtained in this work. It is postulated that part at least of the form of the enzyme that is activated by magnesium is free to diffuse back and forth through pores in the nuclear membrane, whereas this is considered not to be possible for the form of the enzyme that is insensitive to magnesium as a result of the firm binding of the latter to nuclear substance.     

Experience of an interactive program in undergraduate investigative practicals

A microcomputer program is described which has been used by first-year undergraduates throughout an investigation in microbiology, totalling five hours of practical work. The computer is used by students to check their suggestions of possible lines of investigation, the results of practical procedures, and the decisions made on the basis of this experimental evidence. The program offers advice when entries which are inadvisable or incorrect are entered thus providing guidance without usurping student control of the investigation. Evidence for the effectiveness of this program is presented and the potential for this type of consultative program in other areas is indicated. Based on this experience, advice is offered on the general design of this type of program.     

A human placental hormone (UTPH) with uterine growth and DNA promoting effects.

A human placental protein previously described is studied in order to expand its biological characterization. Dose-response-curve of its action on uterine growth of prepuberal mice showed to be a significant logarithmic function and this effect is neutralized by its specific antiserum. It is also demonstrated that the uterine growth-promoting effect is not due either to estrogen, protein-bound estrogen, progesterone or chorionic gonadotrophin. Experimental evidence is given to demonstrate that the mechanism of action of this placental protein is to stimulate the synthesis and to increase the concentration of DNA in uterine tissue, differing then in this respect from the effect of estrogen. Previous work has shown that this placental protein is present in full-term placentas within similar ranges as HCG and HPL. It is also detected in blood during pregnancy and acts biologically in at least two target organs: uterus and mammary gland. Therefore the name of uterotrophic placental protein (UTPH) has been suggested for this substance.     

An Atg13 protein-mediated self-association of the Atg1 protein kinase is important for the induction of autophagy

Autophagy pathways in eukaryotic cells mediate the turnover of a diverse set of cytoplasmic components, including damaged organelles and abnormal protein aggregates. Autophagy-mediated degradation is highly regulated, and defects in these pathways have been linked to a number of human disorders. The Atg1 protein kinase appears to be a key site of this control and is targeted by multiple signaling pathways to ensure the appropriate autophagic response to changing environmental conditions. Despite the importance of this kinase, relatively little is known about the molecular details of Atg1 activation. In this study we show that Atg13, an evolutionarily conserved regulator of Atg1, promotes the formation of a specific Atg1 self-interaction in the budding yeast, Saccharomyces cerevisiae. The appearance of this Atg1-Atg1 complex is correlated with the induction of autophagy, and conditions that disrupt this complex result in diminished levels of both autophagy and Atg1 kinase activity. Moreover, the addition of a heterologous dimerization domain to Atg1 resulted in elevated kinase activity both in vivo and in vitro. The formation of this complex appears to be an important prerequisite for the subsequent autophosphorylation of Thr-226 in the Atg1 activation loop. Previous work indicates that this modification is necessary and perhaps sufficient for Atg1 kinase activity. Interestingly, this Atg1 self-association does not require Atg17, suggesting that this second conserved regulator might activate Atg1 in a manner mechanistically distinct from that of Atg13. In all, this work suggests a model whereby this self-association stimulates the autophosphorylation of Atg1 within its activation loop.     

ON THE OCCURRENCE IN ENGLAND OF THE PEAR FRUIT SAW-FLY, HOPLOCAMPA BEE VIS KLUG

Larvae of Hoplocampa brevis Klug., the Pear Fruit Saw-fly, have been found attacking pears in two gardens in Cambridge. In some continental countries this is a serious pest of pears; this is the first record of damage by this pest in England.
A preliminary study of the biology of this species has been made, and the egg, larval stages and cocoon have been briefly described.
The writer is indebted to Dr H. W. Miles for the identification of the saw-fly and for helpful criticism.     

Conformational Exchange in a Membrane Transport Protein Is Altered in Protein Crystals

Successful macromolecular crystallography requires solution conditions that may alter the conformational sampling of a macromolecule. Here, site-directed spin labeling is used to examine a conformational equilibrium within BtuB, the Escherichia coli outer membrane transporter for vitamin B₁₂. Electron paramagnetic resonance (EPR) spectra from a spin label placed within the N-terminal energy coupling motif (Ton box) of BtuB indicate that this segment is in equilibrium between folded and unfolded forms. In bilayers, substrate binding shifts this equilibrium toward the unfolded form; however, EPR spectra from this same spin-labeled mutant indicate that this unfolding transition is blocked in protein crystals. Moreover, crystal structures of this spin-labeled mutant are consistent with the EPR result. When the free energy difference between substates is estimated from the EPR spectra, the crystal environment is found to alter this energy by 3 kcal/mol when compared to the bilayer state. Approximately half of this energy change is due to solutes or osmolytes in the crystallization buffer, and the remainder is contributed by the crystal lattice. These data provide a quantitative measure of how a conformational equilibrium in BtuB is modified in the crystal environment, and suggest that more-compact, less-hydrated substates will be favored in protein crystals.     

西藏雅鲁藏布江中游斑头雁的越冬种群数量、分布和活动区

斑头雁(Anser indicus)是高致病性禽流感病毒的易感鸟类和潜在的传播源,在禽流感防控中占有重要地位。有关斑头雁的越冬种群现状缺乏研究。2009年1月,在雅鲁藏布江中游共统计到越冬斑头雁44657只,主要分布于一江两河地带的林周县、日喀则市、白朗县、拉孜县、江孜县和贡嘎县境内。这一统计数据远高于1990年代的统计数量,由此可将其全球种群数量估计值由5.2-6万只修正为至少7万只。越冬斑头雁的平均集群大小为(208±262)只(n=215),主要栖息环境包括冬小麦田、冬歇期农田、河流湖泊和沼泽湿地,其中在冬歇期农田中统计到的数量占72.1%。2006-2008年,对青海湖繁殖斑头雁的卫星跟踪表明,其越冬地点主要位于西藏雅鲁藏布江流域、拉萨河流域和印度,越冬期为11月至翌年的3月份,在越冬地平均停留(108±30)d,越冬活动区大小为(122.22±124.94)km2(n=3)。受西藏独特的宗教传统和农耕畜牧结合的生产方式的影响,越冬斑头雁经常与大量家畜和水鸟混杂在农田觅食,在禽流感疫情防控时应引起重视。