Lifetime prevalence of homelessness in housed people aged 55-79 years in England: its childhood correlates and association with mortality over 10 years of follow-up.

OBJECTIVES: Since 2010, the number of homeless people in the UK has increased, and homelessness in its different types has become a major public health problem. Housed older people with past experience of homelessness are an understudied population that can provide valuable insight into this problem. For this reason, we examined the lifetime prevalence of homelessness and its associations with childhood adversity and mortality in a national sample of older people. STUDY DESIGN: This is a longitudinal cohort study. METHODS: We studied 6649 housed individuals aged 55-79 years in 2007 from the English Longitudinal Study of Ageing (ELSA). We used logistic regression to model the association between adverse childhood experiences (ACE) and lifetime experience of homelessness (ever been homeless for ≥1 months) and Cox proportional hazards regression to model the prospective association between lifetime experience of homelessness and mortality. RESULTS: We identified 107 participants with lifetime experience of homelessness. We found a strong graded association between the number of ACE and lifetime experience of homelessness; participants with two ACE had 5.35 (95% confidence interval [CI]: 3.17-9.05) times greater odds of having experienced homelessness than those reporting none. Most ACE were individually associated with lifetime homelessness, but fewer remained so in the mutually adjusted model. Participants with lifetime experience of homelessness had 1.55 (95% CI: 1.01-2.37) times greater risk of mortality over a 10-year follow-up and after adjustment for covariates. CONCLUSIONS: Exposure to childhood adversity is associated with increased risk of experiencing homelessness. Older housed people with past experience of homelessness are at increased risk of mortality.

Association of depression symptoms with receipt of healthcare provider advice on physical activity among US adults.

BACKGROUND: Given the high burden and prevalence of depression, various guidelines underscore the role of healthcare providers in supplying advice on physical activity (PA) as a potential modifying factor influencing the incidence and severity of depressive symptoms in adults. We aimed to investigate the extent to which healthcare providers provide PA advice to adults with depressive symptoms in the US. METHODS: Data on adults aged 20-64 years (n = 4971) in the National Health and Nutrition Examination Study between 2011 and 2016 were analysed. Depressive symptoms were assessed using the Patient Health Questionnaire and response options were categorised as "none or minimal", "mild", "moderate-severe". Receipt of PA advice from a healthcare provider was self-reported. We restricted our study sample to adults free from chronic diseases. RESULTS: Higher odds of receiving advice to exercise were reported among adults with mild (OR = 1.7, 95% CI: 1.3-2.3) and moderate-severe depressive symptoms (OR = 1.7, 95% CI: 1.0-2.8). Furthermore, exercise advice was more commonly reported among adults who were overweight, obese, Hispanic, Asian, being insured with private insurance, with education higher than high school, and had access to a routine place for health care. LIMITATIONS: Social and culutral aspects of overweight/obesity may prohibit generalizations. Cross sectional design does not allow for causal realtionships. CONCLUSIONS: In the US, fewer than one in three adults experiencing symptoms of depression report having received exercise advice from a healthcare provider. Providing such advice may be a sustainable clinical strategy in reducing the incidence and severity of depression symptoms.

Syndemic effects of HIV risk behaviours: results from the NHANES study.

The aim of the present study is to use the syndemic framework to investigate the risk of contracting HIV in the US population. Cross-sectional analyses are from The National Health and Nutrition Examination Survey. We extracted and aggregated data on HIV antibody test, socio-demographic characteristics, alcohol use, drug use, depression, sexual behaviours and sexually transmitted diseases from cycle 2009-2010 to 2015-2016. We carried out weighted regression among young adults (20-39 years) and adults (40-59 years) separately. In total, 5230 men and 5794 women aged 20-59 years were included in the present analyses. In total, 0.8% men and 0.2% women were tested HIV-positive. Each increasing HIV risk behaviour was associated with elevated odds of being tested HIV-positive (1.15, 95% CI 1.15-1.15) among young adults and adults (1.61, 95% CI 1.61-1.61). Multi-faceted, community-based interventions are urgently required to reduce the incidence of HIV in the USA.

Transient activation of human cytomegalovirus lytic gene expression during latency allows cytotoxic T cell killing of latently infected cells.

Human cytomegalovirus (HCMV) latency in the myeloid lineage is maintained by repressive histone modifications around the major immediate early promoter (MIEP), which results in inhibition of the lytic viral life cycle. We now show that pharmacological inhibition of histone deacetylases (HDACs) relieves this repression of the MIEP and induces transient expression of the viral lytic immediate early (IE) antigens but, importantly, not full virus reactivation. In turn, these latently infected cells now become targets for IE-specific cytotoxic T cells (CTLs) which are present at high frequency in all normal healthy HCMV positive carriers but would normally be unable to target latent (lytic antigen-negative) cells. This approach of transiently inducing viral lytic gene expression by HDAC inhibition, in otherwise latently infected cells, offers a window of opportunity to target and purge the latent myeloid cell reservoir by making these normally immunologically undetectable cells visible to pre-existing host immune responses to viral lytic antigens.

Incubation of cocaine cue reactivity associates with neuroadaptations in the cortical serotonin (5-HT) 5-HT2C receptor (5-HT2CR) system.

Intensification of craving elicited by drug-associated cues during abstinence occurs over time in human cocaine users while elevation of cue reactivity ("incubation") is observed in rats exposed to extended forced abstinence from cocaine self-administration. Incubation in rodents has been linked to time-dependent neuronal plasticity in the medial prefrontal cortex (mPFC). We tested the hypothesis that incubation of cue reactivity during abstinence from cocaine self-administration is accompanied by lower potency and/or efficacy of the selective serotonin (5-HT) 5-HT2C​ receptor (5-HT2CR) agonist WAY163909 to suppress cue reactivity and a shift in the subcellular localization profile of the mPFC 5-HT2CR protein. We observed incubation of cue reactivity (measured as lever presses reinforced by the discrete cue complex) between Day 1 and Day 30 of forced abstinence from cocaine relative to sucrose self-administration. Pharmacological and biochemical analyses revealed that the potency of the selective 5-HT2CR agonist WAY163909 to suppress cue reactivity, the expression of synaptosomal 5-HT2CR protein in the mPFC, and the membrane to cytoplasmic expression of the 5-HT2CR in mPFC were lower on Day 30 vs. Day 1 of forced abstinence from cocaine self-administration. Incubation of cue reactivity assessed during forced abstinence from sucrose self-administration did not associate with 5-HT2CR protein expression in the mPFC. Collectively, these outcomes are the first indication that neuroadaptations in the 5-HT2CR system may contribute to incubation of cocaine cue reactivity.

Weight perceptions in a population sample of English adolescents: cause for celebration or concern?

OBJECTIVES: To examine the proportion of normal-weight adolescents who consider themselves to be too heavy (size overestimation), and the proportion of overweight or obese adolescents who consider themselves to be about the right weight or too light (size underestimation), in large population-based samples collected over 8 years in England. METHODS: Data were from the Health Survey for England between 2005 and 2012: an annual survey of households representative of the English population. We analysed data from 4979 adolescents (2668 boys, 2311 girls) aged 13 to 15 years old whose weight status was defined as normal weight or overweight/obese based on body mass index standard deviation scores (BMI-SDS) derived from objective measurements of height and weight and using International Obesity Task Force standards. Weight perception was based on the adolescent's choice from the following descriptors: 'about the right weight', 'too heavy' or 'too light'. RESULTS: The majority of normal-weight adolescents (83% of boys, 84% of girls) correctly identified themselves as 'about the right weight'. Overestimation was uncommon, with only 7% of normal-weight teens (4% of boys, 11% of girls) identifying themselves as 'too heavy'. In contrast, only 60% of overweight/obese adolescents (53% of boys, 68% of girls) correctly identified themselves as 'too heavy', whereas 39% (47% of boys, 32% of girls) underestimated, identifying themselves as 'about the right weight' or 'too light'. There were no significant changes in BMI-SDS or body size estimation over time (2005-2012). CONCLUSIONS: Overestimation of body weight among normal-weight adolescents is relatively uncommon; potentially a cause for celebration. However, almost half of boys and a third of girls with a BMI placing them in the overweight or obese BMI range perceived themselves to be about the right weight. Lack of awareness of excess weight among overweight and obese adolescents could be a cause for concern.

Perceived weight discrimination in England: a population-based study of adults aged ⩾50 years.

BACKGROUND: Despite a wealth of experimental studies on weight bias, little is known about weight discrimination at the population level. This study examined the prevalence and socio-demographic correlates of perceived weight discrimination in a large population-based sample of older adults. METHODS: Data were from 5307 adults in the English Longitudinal Study of Ageing; a population-based cohort of men and women aged ⩾50 years. Weight discrimination was reported for five domains (less respect/courtesy; treated as less clever; poorer treatment in medical settings; poorer service in restaurants/stores; threatened/harassed) at wave 5 (2010-2011). Height and weight were measured at wave 4 (2008-2009). We used logistic regression to test the odds of weight discrimination in relation to weight status, age, sex, wealth, education and marital status. RESULTS: Perceived weight discrimination in any domain was reported by 4.6% of participants, ranging from 0.8% in the normal-weight participants through 0.9, 6.7, 24.2 and 35.1% in individuals who were overweight or met criteria for class I, II and III obesity. Overall, and in each situation, odds of perceived weight discrimination were higher in younger and less wealthy individuals. There was no interaction between weight status and any socio-demographic variable. Relative to normal-weight participants, odds ratios for any perceived weight discrimination were 1.13 (95% confidence interval 0.53-2.40) in those who were overweight, 8.86 (4.65-16.88) in those with class I obesity, 35.06 (18.30-67.16) in class II obese and 56.43 (27.72-114.87) in class III obese. CONCLUSIONS: Our results indicate that rates of perceived weight discrimination are comparatively low in individuals who are overweight or have class I obesity, but for those with class II/III obesity, >10% had experienced discrimination in each domain, and >20% had been treated with less respect or courtesy. These findings have implications for public policy and highlight the need for effective interventions to promote equality.

Diverse specificities, phenotypes, and antiviral activities of cytomegalovirus-specific CD8+ T cells.

UNLABELLED: CD8(+) T cells specific for pp65, IE1, and IE2 are present at high frequencies in human cytomegalovirus (HCMV)-seropositive individuals, and these have been shown to have phenotypes associated with terminal differentiation, as well as both cytokine and proliferative dysfunctions, especially in the elderly. However, more recently, T cell responses to many other HCMV proteins have been described, but little is known about their phenotypes and functions. Consequently, in this study, we chose to determine the diversity of HCMV-specific CD8(+) T cell responses to the products of 11 HCMV open reading frames (ORFs) in a cohort of donors aged 20 to 80 years old as well as the ability of the T cells to secrete gamma interferon (IFN-γ). Finally, we also tested their functional antiviral capacity using a novel viral dissemination assay. We identified substantial CD8(+) T cell responses by IFN-γ enzyme-linked immunospot (ELISPOT) assays to all 11 of these HCMV proteins, and across the cohort, individuals displayed a range of responses, from tightly focused to highly diverse, which were stable over time. CD8(+) T cell responses to the HCMV ORFs were highly differentiated and predominantly CD45RA(+), CD57(+), and CD28(-), across the cohort. These highly differentiated cells had the ability to inhibit viral spread even following direct ex vivo isolation. Taken together, our data argue that HCMV-specific CD8(+) T cells have effective antiviral activity irrespective of the viral protein recognized across the whole cohort and despite viral immune evasion. IMPORTANCE: Human cytomegalovirus (HCMV) is normally carried without clinical symptoms and is widely prevalent in the population; however, it often causes severe clinical disease in individuals with compromised immune responses. HCMV is never cleared after primary infection but persists in the host for life. In HCMV carriers, the immune response to HCMV includes large numbers of virus-specific immune cells, and the virus has evolved many mechanisms to evade the immune response. While this immune response seems to protect healthy people from subsequent disease, the virus is never eliminated. It has been suggested that this continuous surveillance by the immune system may have deleterious effects in later life. The study presented in this paper examined immune responses from a cohort of donors and shows that these immune cells are effective at controlling the virus and can overcome the virus' lytic cycle immune evasion mechanisms.

Investigation of the cutaneous response to recall antigen in humans in vivo.

In this paper we provide a detailed description of an experimental method for investigating the induction and resolution of recall immune response to antigen in humans in vivo. This involves the injection of tuberculin purified protein derivative (PPD) into the skin, followed by inducing suction blisters at the site of injection, from which leucocytes and cytokines that are involved in the response can be isolated and characterized. Using this technique we found that although the majority of CD4(+) T cells in the skin that are present early in the response express cutaneous lymphocyte antigen (CLA), the expression of this marker is reduced significantly in later phases. This may enable these cells to leave the skin during immune resolution. Furthermore, interleukin (IL)-2 production can be detected both in CD4(+) T cells and also in the blister fluid at the peak of the response at day 7, indicating that mediators found in the blister fluid are representative of the cytokine microenvironment in vivo. Finally, we found that older humans have defective ability to respond to cutaneous PPD challenge, but this does not reflect a global immune deficit as they have similar numbers of circulating functional PPD-specific CD4(+) T cells as young subjects. The use of the blister technology enables further characterization of the skin specific defect in older humans and also general mechanisms that govern immune regulation in vivo.

Structural studies on the co-chaperone Hop and its complexes with Hsp90.

The tetratricopeptide repeat domain (TPR)-containing co-chaperone Hsp-organising protein (Hop) plays a critical role in mediating interactions between Heat Shock Protein (Hsp)70 and Hsp90 as part of the cellular assembly machine. It also modulates the ATPase activity of both Hsp70 and Hsp90, thus facilitating client protein transfer between the two. Despite structural work on the individual domains of Hop, no structure for the full-length protein exists, nor is it clear exactly how Hop interacts with Hsp90, although it is known that its primary binding site is the C-terminal MEEVD motif. Here, we have undertaken a biophysical analysis of the structure and binding of Hop to Hsp90 using a variety of truncation mutants of both Hop and Hsp90, in addition to mutants of Hsp90 that are thought to modulate the conformation, in particular the N-terminal dimerisation of the chaperone. The results establish that whilst the primary binding site of Hop is the C-terminal MEEVD peptide of Hsp90, binding also occurs at additional sites in the C-terminal and middle domain. In contrast, we show that another TPR-containing co-chaperone, CyP40, binds solely to the C-terminus of Hsp90. Truncation mutants of Hop were generated and used to investigate the dimerisation interface of the protein. In good agreement with recently published data, we find that the TPR2a domain that contains the Hsp90-binding site is also the primary site for dimerisation. However, our results suggest that residues within the TPR2b may play a role. Together, these data along with shape reconstruction analysis from small-angle X-ray scattering measurements are used to generate a solution structure for full-length Hop, which we show has an overall butterfly-like quaternary structure. Studies on the nucleotide dependence of Hop binding to Hsp90 establish that Hop binds to the nucleotide-free, 'open' state of Hsp90. However, the Hsp90-Hop complex is weakened by the conformational changes that occur in Hsp90 upon ATP binding. Together, the data are used to propose a detailed model of how Hop may help present the client protein to Hsp90 by aligning the bound client on Hsp70 with the middle domain of Hsp90. It is likely that Hop binds to both monomers of Hsp90 in the form of a clamp, interacting with residues in the middle domain of Hsp90, thus preventing ATP hydrolysis, possibly by the prevention of association of N-terminal and middle domains in individual Hsp90 monomers.

Mechanistic studies on Hsp90 inhibition by ansamycin derivatives.

Heat shock protein 90 (Hsp90) is a molecular chaperone that is required for the maturation and activation of a number of client proteins, many of which are involved in cancer development. The ansamycin family of natural products and their derivatives, such as geldanamycin (GA), are well-known inhibitors of the essential ATPase activity of Hsp90. Despite structural studies on the complexes of ansamycin derivatives with the ATPase domain of Hsp90, certain aspects of their inhibitory mechanism remain unresolved. For example, it is known that GA in solution exists in an extended conformation with a trans amide bond; however, it binds to Hsp90 in a significantly more compact conformation with a cis amide bond. GA and its derivatives have been shown to bind to Hsp90 with low micromolar affinity in vitro, in contrast to the low nanomolar anti-proliferative activity that these drugs exhibit in vivo. In addition, they show selectivity towards tumour cells. We have studied both the equilibrium binding, and the association and dissociation kinetics of GA derivative, 17-DMAG, and the fluorescently labelled analogue BDGA to both wild-type and mutant Hsp90. The mutants were made in order to test the hypothesis that conserved residues near the ATP-binding site may catalyse the trans-cis isomerisation of GA. Our results show that Hsp90 does not catalyse the trans-cis isomerisation of GA, and suggests that there is no isomerisation step before binding to Hsp90. Experiments with BDGA measured over a wide range of conditions, in the absence and in the presence of reducing agents, confirm recent studies that have suggested that the reduced dihydroquinone form of the drug binds to Hsp90 considerably more tightly than the non-reduced quinone species.

A comparison of experimental and computational methods for mapping the interactions present in the transition state for folding of FKBP12.

The folding pathway of FKBP12, a 107 residue α/ß protein, has been characterised in detail using a combination of experimental and computational techniques. FKBP12 follows a two-state model of folding in which only the denatured and native states are significantly populated; no intermediate states are detected. The refolding rate constant in water is 4 s(-1) at 25 °C. Two different experimental strategies were employed for studying the transition state for folding. In the first case, a non-mutagenic approach was used and the unfolding and refolding of the wild-type protein measured as a function of experimental conditions such as temperature, denaturant, ligand and trifluoroethanol (TFE) concentration. These data suggest a compact transition state relative to the unfolded state with some 70% of the surface area buried. The ligand-binding site, whichis mainly formed by two long loops, is largely unstructured in the transition state. TFE experiments suggest that the α-helix may be formed in the transition state. The second experimental approach involved using protein engineering techniques with φ-value analysis. Residue-specific information on the structure and energetics of the transition state can be obtained by this method. 34 mutations were made at sites throughout the protein to probe the extent of secondary and tertiary structure in the transition state. In contrast to some other proteins of this size, no element of structure is fully formed in the transition state, instead, the transition state is similar to that found for smaller, single-domain proteins, such as chymotrypsin inhibitor 2 and the SH3 domainfrom α-spectrin. For FKBP12, the central three strands of the ß-sheet (2, 4 and 5), comprise the most structured region of the transition state. In particular Val 101, which is one of the most highly buried residues and located in the middle of the central ß-strand,makes approximately 60% of its native interactions. The outer ß-strands, and the ends of the central ß-strands are formed to a lesser degree. The short α-helix is largely unstructured in the transition state as are the loops. The data are consistent with a nucleation-condensation model of folding, the nucleus of which is formed by side chains within ß-strands 2, 4 and 5 and the C-terminus of the α-helix. These residues are distant in the primary sequence, demonstrating the importance of tertiary interactions in the transition state. High-temperature molecular dynamic simulations on the unfoldingpathway of FKBP12 are in good agreement with the experimental results.

Non-chondritic distribution of the highly siderophile elements in mantle sulphides.

The abundances of highly siderophile (iron-loving) elements (HSEs) in the Earth's mantle provide important constraints on models of the Earth's early evolution. It has long been assumed that the relative abundances of HSEs should reflect the composition of chondritic meteorites--which are thought to represent the primordial material from which the Earth was formed. But the non-chondritic abundance ratios recently found in several types of rock derived from the Earth's mantle have been difficult to reconcile with standard models of the Earth's accretion, and have been interpreted as having arisen from the addition to the primitive mantle of either non-chondritic extraterrestrial material or differentiated material from the Earth's core. Here we report in situ laser-ablation analyses of sulphides in mantle-derived rocks which show that these sulphides do not have chondritic HSE patterns, but that different generations of sulphide within single samples show extreme variability in the relative abundances of HSEs. Sulphides enclosed in silicate phases have high osmium and iridium abundances but low Pd/Ir ratios, whereas pentlandite-dominated interstitial sulphides show low osmium and iridium abundances and high Pd/Ir ratios. We interpret the silicate-enclosed sulphides as the residues of melting processes and interstitial sulphides as the crystallization products of sulphide-bearing (metasomatic) fluids. We suggest that non-chondritic HSE patterns directly reflect processes occurring in the upper mantle--that is, melting and sulphide addition via metasomatism--and are not evidence for the addition of core material or of 'exotic' meteoritic components.

Does trifluoroethanol affect folding pathways and can it be used as a probe of structure in transition states?

Nonaqueous co-solvents, particularly 2,2,2-trifluoroethanol (TFE), have been used as tools to study protein folding. By analyzing FKBP12, an alpha/beta-protein that folds with two-state kinetics, we have been able to address three key questions concerning the use of TFE. First, does TFE perturb the folding pathway? Second, can the observed changes in the rate of folding and unfolding in TFE be attributed to a change in free energy of a single state? Finally, can TFE be used to infer information on secondary structure formation in the transition state? Protein engineering experiments on FKBP12, coupled with folding and unfolding experiments in 0% and 9.6% TFE, conclusively show that TFE does not perturb the folding pathway of this protein. Our results also suggest that the changes in folding and unfolding rates observed in 9.6% TFE are due to a global effect of TFE on the protein, rather than the stabilization of any elements of secondary structure in the transition state. Thus, studies with TFE and other co-solvents can be accurately interpreted only when combined with other techniques.

Folding pathway of FKBP12 and characterisation of the transition state.

The folding pathway of human FKBP12, a 12 kDa FK506-binding protein (immunophilin), has been characterised. Unfolding and refolding rate constants have been determined over a wide range of denaturant concentrations and data are shown to fit to a two-state model of folding in which only the denatured and native states are significantly populated, even in the absence of denaturant. This simple model for folding, in which no intermediate states are significantly populated, is further supported from stopped-flow circular dichroism experiments in which no fast "burst" phases are observed. FKBP12, with 107 residues, is the largest protein to date which folds with simple two-state kinetics in water (kF=4 s(-1)at 25 degrees C). The topological crossing of two loops in FKBP12, a structural element suggested to cause kinetic traps during folding, seems to have little effect on the folding pathway. The transition state for folding has been characterised by a series of experiments on wild-type FKBP12. Information on the thermodynamic nature of, the solvent accessibility of, and secondary structure in, the transition state was obtained from experiments measuring the unfolding and refolding rate constants as a function of temperature, denaturant concentration and trifluoroethanol concentration. In addition, unfolding and refolding studies in the presence of ligand provided information on the structure of the ligand-binding pocket in the transition state. The data suggest a compact transition state relative to the unfolded state with some 70 % of the surface area buried. The ligand-binding site, which is formed mainly by two loops, is largely unstructured in the transition state. The trifluoroethanol experiments suggest that the alpha-helix may be formed in the transition state. These results are compared with results from protein engineering studies and molecular dynamics simulations (see the accompanying paper).

Mapping the interactions present in the transition state for unfolding/folding of FKBP12.

The structure of the transition state for folding/unfolding of the immunophilin FKBP12 has been characterised using a combination of protein engineering techniques, unfolding kinetics, and molecular dynamics simulations. A total of 34 mutations were made at sites throughout the protein to probe the extent of secondary and tertiary structure in the transition state. The transition state for folding is compact compared with the unfolded state, with an approximately 30 % increase in the native solvent-accessible surface area. All of the interactions are substantially weaker in the transition state, as probed by both experiment and molecular dynamics simulations. In contrast to some other proteins of this size, no element of structure is fully formed in the transition state; instead, the transition state is similar to that found for smaller, single-domain proteins, such as chymotrypsin inhibitor 2 and the SH3 domain from alpha-spectrin. For FKBP12, the central three strands of the beta-sheet, beta-strand 2, beta-strand 4 and beta-strand 5, comprise the most structured region of the transition state. In particular Val101, which is one of the most highly buried residues and located in the middle of the central beta-strand, makes approximately 60 % of its native interactions. The outer beta-strands and the ends of the central beta-strands are formed to a lesser degree. The short alpha-helix is largely unstructured in the transition state, as are the loops. The data are consistent with a nucleation-condensation model of folding, the nucleus of which is formed by side-chains within beta-strands 2, 4 and 5, and the C terminus of the alpha-helix. The precise residues involved in the nucleus differ in the two simulated transition state ensembles, but the interacting regions of the protein are conserved. These residues are distant in the primary sequence, demonstrating the importance of tertiary interactions in the transition state. The two independently derived transition state ensembles are structurally similar, which is consistent with a Bronsted analysis confirming that the transition state is an ensemble of states close in structure.

Elemental composition of human milk from mothers of premature and full-term infants during the first 3 months of lactation.

To examine longitudinal and gestational effects of mineral content in human milk, we analyzed human milk from lactating mothers of premature (PRT, n = 24, < 2000 g birth weight, < 37 wk gestation) and full-term (FT, n = 19, > 2500 g, 39-41 wk gestation), living in Newfoundland, Canada. Samples were collected once a week for 8 wk with one final sample collected at 3 mo. Milk samples collected in acid-washed containers were wet ashed with concentrated HNO3, and barium, cadmium, calcium, cesium, cobalt, copper, cerium, lanthanum, magnesium, manganese, molybdenum, nickel, lead, rubidium, tin, strontium, and zinc were measured using inductively coupled plasma-mass spectrometry. Data were analyzed using standard multiple-regression procedures with correlated data analyses to take account of the relationship between successive weeks. Results indicated lower Ca and Pb in PRT milk. Calcium was the only nutritionally significant element to differ between groups. Molybdenum in both PRT and FT milk showed a definite decrease with time, suggesting that the Mo content in milk is homeostatically regulated. However, Ce, La, Ba, and Sn did not display any pattern indicative of biological regulation and potential human requirement.

How do small single-domain proteins fold?

Many small, monomeric proteins fold with simple two-state kinetics and show wide variation in folding rates, from microseconds to seconds. Thus, stable intermediates are not a prerequisite for the fast, efficient folding of proteins and may in fact be kinetic traps and slow the folding process. Using recent studies, can we begin to search for trends which may lead to a better understanding of the protein folding process?

Context-dependent nature of destabilizing mutations on the stability of FKBP12.

The context-dependent nature in which mutations affect protein stability was investigated using the FK506-binding protein, FKBP12. Thirty-four mutations were made at sites throughout the protein, including residues located in the hydrophobic core, the beta-sheet, and the solvent-exposed face of the alpha-helix. Urea-induced denaturation experiments were used to measure the change in stability of the mutants relative to that of the wild type (Delta DeltaGU-F). The results clearly show that the extent of destabilization, or stabilization, is highly context-dependent. Correlations were sought in order to link Delta DeltaGU-F to various structural parameters. The strongest correlation found was between Delta DeltaGU-F and N, the number of methyl(ene) groups within a 6 A radius of the group(s) deleted. For mutations of buried hydrophobic residues, a correlation coefficient of 0.73 (n = 16,where n is the number of points) was obtained. This increased to 0.81 (n = 24) on inclusion of mutations of partially buried hydrophobic residues. These data could be superimposed on data obtained for other proteins for which similarly detailed studies have been performed. Thus, the contribution to stability from hydrophobic side chains, independent of the extent to which a side chain is buried, can be estimated quantitatively using N. This correlation appears to be a general feature of all globular proteins. The effect on stability of mutating polar and charged residues in the alpha-helix and beta-sheet was also found to be highly context-dependent. Previous experimental and statistical studies have shown that specific side chains can stabilize the N-caps of alpha-helices in proteins. Substitutions of Ile56 to Thr and Asp at the N-cap of the alpha-helix of FKBP12, however, were found to be highly destabilizing. Thus, the intrinsic propensities of an amino acid for a particular element of secondary structure can easily be outweighed by tertiary packing factors. This study highlights the importance of packing density in determining the contribution of a residue to protein stability. This is the most important factor that should be taken into consideration in protein design.