Hair cortisol as a risk marker for increased depressive symptoms among older adults during the COVID-19 pandemic.

Determining pre-existing biological risk markers of incident depression and other mental health sequelae after exposure to a new stressor would help identify vulnerable individuals and mechanistic pathways. This study investigated primarily whether hair cortisol predicted elevated depressive symptoms in middle-aged and older adults during the COVID-19 pandemic, 6 years later. A secondary aim was to deduce whether any association differed by sex. METHODS: We studied 1025 adults aged 50 and older (75% female) as part of The Irish Longitudinal Study on Ageing. Hair cortisol samples were collected at 2014 (Wave 3) and depressive symptoms were assessed using the 8-item Center for Epidemiological Studies Depression Scale in 2014 (Wave 3), 2016 (Wave 4), 2018 (Wave 5) and again in 2020 as part of TILDA's COVID-19 Study. Hierarchical mixed effects logistic regression models were applied to investigate the association between cortisol levels and clinically significant depressive symptoms before and during the COVID-19 pandemic. RESULTS: In a full covariate adjusted model there was a significant interaction between cortisol and wave on depressive symptoms (χ2 = 8.5, p = .03). Cortisol was positively and significantly associated with elevated depressive symptoms during the COVID-19 Study (OR =1.3, 95% CI 1.11, 1.56, p = .003), and was associated with an increased likelihood of reporting clinically significant depressive symptoms during first year of the COVID-19 pandemic, when compared with before, OR = 1.4, 95% CI 1.05, 1.9, p = .015. There was no evidence of effect modification by sex. CONCLUSIONS: Higher hair cortisol, assessed 6 years previously, predicted clinically significant depressive symptoms among middle-aged and older adults during (but not before) the pandemic. Findings suggest a biological phenotype which denotes increased susceptibility to the negative impact of environmental stress on psychological health.

Change in perceived stress and 2-year change in cognitive function among older adults: The Irish Longitudinal Study on Ageing.

Prolonged or severe stress can adversely affect older adults' cognitive function, but population-based studies investigating this relationship over time are rare. Previous studies have largely focused on stress either evaluated at a single time point or linked to specific life events. This study aimed to investigate whether a change in perceived stress over 2 years predicts a change in cognitive performance over the same time period in a population-based sample of older adults. Data from the first 2 waves of The Irish Longitudinal Study on Ageing were analyzed. Five thousand and seventy adults aged 50 and older completed the 4-item Perceived Stress Scale and measures of verbal fluency, immediate and delayed word recall 2 years apart. A first differences regression model revealed that the change in stress over 2 years was inversely associated with a change in immediate word recall performance, even after adjustment for change in possible confounders (B = -0.030, 95% CI [-.056, -.004], p < .05). No association was observed for delayed recall or verbal fluency performance. Change in perceived stress is inversely correlated with change in immediate recall, even over a short period. Stress modifying interventions could potentially reduce the risk of cognitive decline associated with ageing.

Is a stepdown unit safe for patients with mild traumatic intracranial hemorrhages?

PURPOSE: Traumatic brain injuries (TBIs) are a major source of disability in the United States. The ideal unit in the hospital for patients with mild traumatic intracranial hemorrhages (ICHs) has not been elucidated. We sought to investigate whether patients treated in the surgical stepdown area had worse outcomes than those treated in the surgical ICU. METHODS: We compared patients with ICHs and a Glasgow Coma Scale (GCS) upon admission of 14 or 15 who went to the ICU to those who went to the stepdown area from April 2014 to November 2016. We compared age, gender, Injury Severity Score (ISS), admission GCS (14 or 15), operative intervention, discharge destination, hospital length of stay (HLOS), mortality, and cost between these two groups. RESULTS: Patients admitted to the ICU had a significantly longer HLOS. Admission costs for patients admitted to ICU were also significantly higher than their stepdown area counterparts. This was true for both total charges (p = 0.0001) and for net revenue (p = 0.002) (Table 2). There was no statistically significant difference in mortality, operative intervention, or discharge destination. CONCLUSION: A surgical stepdown unit can be a safe disposition for patients with mild traumatic ICHs and represents an effective use of hospital resources.

Non-Dietary Correlates and Determinants of Plasma Lutein and Zeaxanthin Concentrations in the Irish Population.

OBJECTIVE: To investigate non-dietary correlates and determinants of plasma lutein (L) and zeaxanthin (Z) concentrations in The Irish Longitudinal Study on Ageing (TILDA) sample. DESIGN: Cross-sectional study. SETTING: Community dwelling adults in the Republic of Ireland (ROI). PARTICIPANTS: 3,681 participants aged 50 years and older. MEASUREMENTS: TILDA is a nationally representative prospective cohort study of community dwelling adults aged 50 years and over in the ROI. Demographic and health variables were collected during a face-to-face interview carried out in the home (n=8175), and a substantial proportion of these (n=5035; 62%) also attended a study visit in a health assessment centre. Blood samples collected at baseline (wave 1, the subject of the current study), were analysed for plasma concentrations of L and Z by reversed-phase high performance liquid chromatography, and macular pigment (MP) optical density was also measured (using customized heterochromatic flicker photometry). RESULTS: After excluding participants with eye disease, data from 3,681 participants were available for analysis. For this group of participants, plasma L and Z were inversely and significantly associated with body mass index (BMI), and were positively and significantly associated with MP, total cholesterol, high-density lipoprotein (HDL) and low-density lipoprotein (LDL) (p<0.001, for all). Plasma L and Z were significantly lower in males, current smokers, participants reporting less physical exercise, and participants reporting lower levels of education (p<0.05, for all). Plasma L was significantly higher in participants reporting a family history of age-related macular degeneration (AMD) (p=0.001), and in the group of ≥75 years old (p<0.05). For each of these variables, the significant associations remained after controlling for other potential confounding variables. CONCLUSION: The findings of this large study indicate that plasma concentrations of L and Z were lower in association with indicators of a poor lifestyle (high BMI, tobacco use, and less physical exercise) and in association with lower education, indicating that modifying lifestyle in a positive way is likely to be reflected in higher concentrations of plasma carotenoids, with consequential and putative health benefits.

Growth of a progesterone receptor-positive meningioma in a female patient with congenital adrenal hyperplasia.

Meningioma growth has been previously described in patients receiving oestrogen/progestogen therapy. We describe the clinical, radiological, biochemical and pathologic findings in a 45-year-old woman with congenital adrenal hyperplasia secondary to a defect in the 21-hydroxylase enzyme who had chronic poor adherence to glucocorticoid therapy with consequent virilisation. The patient presented with a frontal headache and marked right-sided proptosis. Laboratory findings demonstrated androgen excess with a testosterone of 18.1 nmol/L (0-1.5 nmol) and 17-Hydroxyprogesterone >180 nmol/L (<6.5 nmol/L). CT abdomen was performed as the patient complained of rapid-onset increasing abdominal girth and revealed bilateral large adrenal myelolipomata. MRI brain revealed a large meningioma involving the right sphenoid wing with anterior displacement of the right eye and associated bony destruction. Surgical debulking of the meningioma was performed and histology demonstrated a meningioma, which stained positive for the progesterone receptor. Growth of meningioma has been described in postmenopausal women receiving hormone replacement therapy, in women receiving contraceptive therapy and in transsexual patients undergoing therapy with high-dose oestrogen and progestogens. Progesterone receptor positivity has been described previously in meningiomas. 17-Hydroxyprogesterone is elevated in CAH and has affinity and biological activity at the progesterone receptor. Therefore, we hypothesise that patients who have long-standing increased adrenal androgen precursor concentrations may be at risk of meningioma growth. LEARNING POINTS: Patients with long-standing CAH (particularly if not optimally controlled) may present with other complications, which may be related to long-standing elevated androgen or decreased glucocorticoid levels.Chronic poor control of CAH is associated with adrenal myelolipoma and adrenal rest tissue tumours.Meningiomas are sensitive to endocrine stimuli including progesterone, oestrogen and androgens as they express the relevant receptors.

The improved accuracy of planar bone scintigraphy by adding single photon emission computed tomography (SPECT-CT) to detect skeletal metastases from prostate cancer.

BACKGROUND: Planar bone scintigraphy (PBS) is a standard radiological technique to detect skeletal metastases from prostate cancer (PC), the addition of SPECT-CT to PBS improves its diagnostic accuracy. The aim of this study was to assess the additional value of targeted SPECT-CT with PBS in detecting skeletal metastasis form prostate cancer, considering resource implications in an Irish hospital setting. METHODS: 54 PC patients with increased radiotracer uptake on PBS were retrospectively recruited from 2012 to 2013. All underwent targeted evaluation with SPECT-CT. PBS and SPECT-CT images were reviewed by two nuclear medicine radiologists and reported independently. The final diagnosis was made based on the CT finding corresponding to the area of radiotracer uptake. RESULTS: The mean age was 70.9 years (48-88 years) and median PSA at presentation was 13.9 ng/ml (4.2-215 ng/ml). 68.5 % (n = 37) men received treatment for PC while 31.5 % (n = 17) patients had not received treatment prior to PBS. 164 areas of increased radiotracer uptake were identified on PBS; 13 areas were characterised as metastatic on SPECT-CT; iliac bone (n = 3), ribs (n = 1), skull (n = 2), sacrum (n = 1), ischium (n = 1), femur (n = 3), thoracic spine (n = 1) and cervical spine (n = 1). 151 areas were characterised as benign on SPECT-CT. One area of increased radiotracer uptake in the ribs was subsequently described as indeterminate after evaluation with SPECT-CT. CONCLUSION: SPECT-CT improves the diagnostic accuracy of PBS in detecting skeletal metastasis from PC and is superior to PBS alone in differentiating benign from malignant lesions. Notwithstanding resource implications of increased cost, specialist equipment and specialist manpower hours; we recommend the use of SPECT-CT in conjunction with PBS for targeted evaluation of suspicious bony lesions in this cohort of patients.

Sexual risk behavior, alcohol use, and social media use among secondary school students in informal settlements in Cape Town and Port Elizabeth, South Africa.

South Africa's HIV prevalence among young people remains among the highest in the world. A cross-sectional study was carried out in 2012 to estimate prevalences of sexual risk behavior and hazardous alcohol use (HAU) (via the Alcohol Use Disorder Identification Test) as well as to investigate potential associations between these outcomes and social media use. In all, 4485 students (mean age 15.66 years, SD 1.39) at 46 secondary schools in informal settlements in Cape Town and Port Elizabeth completed mobile-phone-assisted, self-administered baseline questionnaires within a cluster-randomized trial. In all, 312 females (12.5 %) and 468 males (23.5 %) screened positive for HAU (AOR = 1.98, 95 % CI 1.69-2.34). 730 males (39.9 %) and 268 females (11.8 %) reported having had two or more partners in the last year (AOR = 3.46, 95 % CI 2.87-4.16). Among females, having a Facebook account was associated with reported multiple partnerships in the last year (AOR = 1.81, 95 % CI 1.19-2.74), age-disparate sex in the last year (AOR = 1.96, 95 % CI 1.16-3.32) and HAU (AOR = 1.97, 95 % CI 1.41-2.74). Using Mxit-a popular mobile instant messaging application-was associated with higher odds of reported multiple partnerships in the last year among both males (AOR = 1.70, 95 % CI 1.35-2.14) and females (AOR = 1.45, 95 % CI 1.07-1.96) and with HAU among both males (AOR = 1.47, 95 % CI 1.14-1.90) and females (AOR = 1.50, 95 % CI 1.18-1.90). Further longitudinal and qualitative research should explore in more depth the observed links between social media and risk behavior.

Prehospital HMG Co-A reductase inhibitor use and reduced mortality in hemorrhagic shock due to trauma.

INTRODUCTION: 3-Hydroxy-3-methyl-glutaryl Co-A reductase inhibitors (HMG Co-A reductase inhibitors, statins) are commonly used medications for the control of serum cholesterol. Recent data suggests that these medications also modify the inflammatory pathways in sepsis, septic shock, and hemorrhagic shock due to ruptured abdominal aortic aneurysms. Statin use in hemorrhagic shock due to trauma, however, has conflicting data, with one study showing improvement, but only in certain subsets of patients. STUDY DESIGN: We retrospectively reviewed the medical records of patients from our institution's trauma registry database from January 2000 to December 2008. We included patients with an age greater than 45 years and an Injury Severity Score (ISS) greater than 15 with evidence of shock as follows: hypotension, elevated serum lactate, base deficit, metabolic acidosis, or objective evidence of end-organ malperfusion. We excluded patients with devastating head injury, patients with pre-existing advance directives directing against life-sustaining measures, patients for whom family or health care proxies withdrew support in 24 h or less, and patients who succumbed to their injuries in the first 24 h in the hospital. We compared age, gender, mortality, statin use, aspirin use, and Sequential Organ Failure Assessment (SOFA) scores. RESULTS: Mortality in the group without prehospital statin use was 38.1% (95% confidence interval [CI]: 28.4-48.8%) and mortality in the group with prehospital statin use was 8.3% (95% CI: 2.13-22.5%, P = 0.0009). The absolute risk reduction was 29.8% and the relative risk reduction was 78.1%. Survivors were statistically significantly younger than nonsurvivors in the group without prehospital statin use, but not in the group with documented prehospital statin use. There was no similar benefit to aspirin use. There were no significant differences in the SOFA scores, hospital length of stay (HLOS), or intensive care unit length of stay (ICU LOS) between statin users and nonusers. CONCLUSIONS: Prehospital HMG Co-A reductase use was associated with improved survival in a population with severe trauma and evidence of ongoing hemorrhagic shock.

Radiological impact of the use of calcium hydroxylapatite dermal fillers.

AIM: To report a case series in which the radiological features of the subcutaneous use of calcium hydroxylapatite (CaHa) dermal fillers are described for the first time. MATERIALS AND METHODS: Five patients with facial hyperattenuating hypermetabolic subcutaneous lesions were identified on 2- [(18)F]-fluoro-2-deoxy-D-glucose (FDG) positron-emission tomography/computed tomography (PET/CT), who gave a history of facial injections to augment physical appearance. Correlation with additional imaging studies was performed. RESULTS: All cases had subcutaneous high attenuation material on CT (range 280-700HU), which was FDG avid on PET, with a standardized uptake value (SUV) range of 2.9-13.4. Magnetic resonance imaging (MRI) demonstrated a heterogeneous intermediate signal intensity subcutaneous lesion with enhancement post-gadolinium in one case. CONCLUSIONS: CaHa dermal filler is hyperattenuating on CT, hypermetabolic on FDG-PET imaging, of intermediate signal intensity on MRI, and is a potential cause of a false-positive imaging study.

Imaging of acute pyelonephritis in the adult.

The diagnosis of acute pyelonephritis in adults is predominantly made by a combination of typical clinical features of flank pain, high temperature and dysuria combined with urinalysis findings of bacteruria and pyuria. Imaging is generally reserved for patients who have atypical presenting features or in those who fail to respond to conventional therapy. In addition, early imaging may be useful in diabetics or immunocompromised patients. In such patients, imaging may not only aid in making the diagnosis of acute pyelonephritis, but more importantly, it may help identify complications such as abscess formation. In this pictorial review, we discuss the role of modern imaging in acute pyelonephritis and its complications. We discuss the growing role of cross-sectional imaging with computed tomography (CT) and novel magnetic resonance imaging (MRI) techniques that may be used to demonstrate both typical as well as unusual manifestations of acute pyelonephritis and its complications. In addition, conditions such as emphysematous and fungal pyelonephritis are discussed.

NMR solution studies of hamster galectin-3 and electron microscopic visualization of surface-adsorbed complexes: evidence for interactions between the N- and C-terminal domains.

Galectin-3, a beta-galactoside binding protein, contains a C-terminal carbohydrate recognition domain (CRD) and an N-terminal domain that includes several repeats of a proline-tyrosine-glycine-rich motif. Earlier work based on a crystal structure of human galectin-3 CRD, and modeling and mutagenesis studies of the closely homologous hamster galectin-3, suggested that N-terminal tail residues immediately preceding the CRD might interfere with the canonical subunit interaction site of dimeric galectin-1 and -2, explaining the monomeric status of galectin-3 in solution. Here we describe high-resolution NMR studies of hamster galectin-3 (residues 1--245) and several of its fragments. The results indicate that the recombinant N-terminal fragment Delta 126--245 (residues 1--125) is an unfolded, extended structure. However, in the intact galectin-3 and fragment Delta 1--93 (residues 94--245), N-terminal domain residues lying between positions 94 and 113 have significantly reduced mobility values compared with those expected for bulk N-terminal tail residues, consistent with an interaction of this segment with the CRD domain. In contrast to the monomeric status of galectin-3 (and fragment Delta 1--93) in solution, electron microscopy of negatively stained and rotary shadowed samples of hamster galectin-3 as well as the CRD fragment Delta 1--103 (residues 104--245) show the presence of a significant proportion (up to 30%) of oligomers. Similar imaging of the N-terminal tail fragment Delta 126--245 reveals the presence of fibrils formed by intermolecular interactions between extended polypeptide subunits. Oligomerization of substratum-adsorbed galectin-3, through N- and C-terminal domain interactions, could be relevant to the positive cooperativity observed in binding of the lectin to immobilized multiglycosylated proteins such as laminin.

Clinical outcomes of aneurysmal subarachnoid hemorrhage patients treated with oral diltiazem and limited intensive care management.

BACKGROUND: Aneurysmal subarachnoid hemorrhage (SAH) patients are frequently treated with prophylactic nimodipine and undergo invasive monitoring of blood pressure and volume status in an intensive care unit (ICU) setting to decrease the incidence of delayed ischemic neurological deficit (DIND) and improve functional outcomes. The goal of this study was to examine the incidence of DIND and poor functional outcomes in a consecutive series of SAH patients treated with a different regimen of prophylactic oral diltiazem and limited use of intensive care monitoring. METHODS: The study involved a consecutive series of 123 aneurysmal SAH patients treated by the senior author who were admitted within 72 hours of hemorrhage and who never received nimodipine or nicardipine. Functional outcomes were graded using the Glasgow Outcome Scale (GOS). RESULTS: Of the 123 patients identified, favorable outcomes (GOS 4 and 5) were achieved in 74.8%. The incidence of DIND was 19.5%. Hypertensive, hypervolemic, hemodilutional (HHH) therapy was used in 10 patients (8.1%) and no patients were treated for DIND by endovascular means. Seven patients (5.7%) had a poor functional outcome or death because of DIND and two of these were related to complications of HHH therapy. These results were compared to contemporary series of SAH patients managed with other treatment protocols. CONCLUSIONS: Functional outcomes of patients treated with a regimen of oral diltiazem, limited use of ICU monitoring and HHH therapy for DIND compare favorably with other contemporary series of SAH patients.

The solution structure of the disulphide-linked homodimer of the human trefoil protein TFF1.

The trefoil factor family protein, TFF1, forms a homodimer, via a disulphide linkage, that has greater activity in wound healing assays than the monomer. Having previously determined a high-resolution solution structure of a monomeric analogue of TFF1, we now investigate the structure of the homodimer formed by the native sequence. The two putative receptor/ligand recognition domains are found to be well separated, at opposite ends of a flexible linker. This contrasts sharply with the known fixed and compact arrangement of the two trefoil domains of the closely related TFF2, and has significant implications for the mechanism of action and functional specificity of the TFF of proteins.

Inhibitory and blocking monoclonal antibody epitopes on merozoite surface protein 1 of the malaria parasite Plasmodium falciparum.

Merozoite surface protein 1 (MSP-1) is a precursor to major antigens on the surface of Plasmodium spp. merozoites, which are involved in erythrocyte binding and invasion. MSP-1 is initially processed into smaller fragments; and at the time of erythrocyte invasion one of these of 42 kDa (MSP-1(42)) is subjected to a second processing, producing 33 kDa and 19 kDa fragments (MSP-1(33) and MSP-1(19)). Certain MSP-1-specific monoclonal antibodies (mAbs) react with conformational epitopes contained within the two epidermal growth factor domains that comprise MSP-1(19), and are classified as either inhibitory (inhibit processing of MSP-1(42) and erythrocyte invasion), blocking (block the binding and function of the inhibitory mAb), or neutral (neither inhibitory nor blocking). We have mapped the epitopes for inhibitory mAbs 12.8 and 12.10, and blocking mAbs such as 1E1 and 7.5 by using site-directed mutagenesis to change specific amino acid residues in MSP-1(19) and abolish antibody binding, and by using PEPSCAN to measure the reaction of the antibodies with every octapeptide within MSP-1(42). Twenty-six individual amino acid residue changes were made and the effect of each on the binding of mAbs was assessed by Western blotting and BIAcore analysis. Individual changes had either no effect, or reduced, or completely abolished the binding of individual mAbs. No two antibodies had an identical pattern of reactivity with the modified proteins. Using PEPSCAN each mAb reacted with a number of octapeptides, most of which were derived from within the first epidermal growth factor domain, although 1E1 also reacted with peptides spanning the processing site. When the single amino acid changes and the reactive peptides were mapped onto the three-dimensional structure of MSP-1(19), it was apparent that the epitopes for the mAbs could be defined more fully by using a combination of both mutagenesis and PEPSCAN than by either method alone, and differences in the fine specificity of binding for all the different antibodies could be distinguished. The incorporation of several specific amino acid changes enabled the design of proteins that bound inhibitory but not blocking antibodies. These may be suitable for the development of MSP-1-based vaccines against malaria.

Mortality in antiepileptic drug development programs.

BACKGROUND: Pooled data from New Drug Applications (NDAs) submitted to the U.S. Food and Drug Administration (FDA) provide an opportunity to study the incidence of and risk factors for rare events. OBJECTIVE: To examine the incidence and causes of mortality in patients with epilepsy participating in clinical trials of antiepileptic drugs (AEDs); and to examine the incidence of and risk factors for sudden unexplained death in such patients. METHODS: Exposure data and death narratives were obtained from the NDAs of five recently reviewed AEDs. Deaths were classified as sudden unexplained, accidental, or other cause using the 1993 Burroughs-Wellcome expert panel criteria, and mortality rates were calculated for each category. Add-on trials were analyzed separately from monotherapy initiation trials. RESULTS: Among 9,144 patients in the add-on trial database, the all-cause and sudden unexplained mortality rates were 9.1 and 3.8 deaths per 1,000 person-years (124 and 52 deaths in 13,617.1 person-years of drug exposure). Sixty-five percent of all deaths were related to the underlying epilepsy. Of the examined risk factors, only age was associated with the incidence of sudden unexplained death. Among 1,293 patients in the monotherapy initiation trials, the all-cause and sudden unexplained mortality rates were 7.1 and 0 deaths per 1,000 person-years (7 and 0 deaths in 982.5 person-years of drug exposure). CONCLUSIONS: A large proportion of the deaths in the add-on cohort was attributable to epilepsy-related causes. Mortality due to sudden death in the add-on cohort falls into the high end of the reported range for patients with epilepsy. The difference in mortality due to sudden death between the add-on and monotherapy initiation cohorts suggests that disease severity is the primary determining factor for risk of sudden unexplained death.

Calcium-induced refolding of the calmodulin V136G mutant studied by NMR spectroscopy: evidence for interaction between the two globular domains.

The Ca(2+) titration of the (15)N-labeled mutant V136G calmodulin has been monitored using (1)H-(15)N HSQC NMR spectra. Up to a [Ca(2+)]/[CaM] ratio of 2, the Ca(2+) ions bind predominantly to sites I and II on the N-domain in contrast with the behavior of the wild-type calmodulin where the C-terminal domain has the higher affinity for Ca(2+). Surprisingly, the Ca(2+)-binding affinity for the N-domain in the mutant calmodulin is greater than that for the N-domain in the wild-type protein. The mutated C-domain is observed as a mixture of unfolded, partially folded (site III occupied), and native-like folded (sites III and IV occupied) conformations, with relative populations dependent on the [Ca(2+)]/[CaM] ratio. The occupancy of site III independently of site IV in this mutant shows that the cooperativity of Ca(2+) binding in the C-domain is mediated by the integrity of the domain structure. Several NH signals from residues in the Ca(2+)-bound N-domain appear as two signals during the Ca(2+) titration indicating separate species in slow exchange, and it can be deduced that these result from the presence and absence of interdomain interactions in the mutant. It is proposed that an unfolded part of the mutated C-domain interacts with sites on the N-domain that normally bind to target proteins. This would also account for the increase in the Ca(2+) affinity for the N-domain in the mutant compared with the wild-type calmodulin. The results therefore show the wide-ranging effects of a point mutation in a single Ca(2+)-binding site, providing details of the involvement of individual residues in the calcium-induced folding reactions.

Expression of deuterium-isotope-labelled protein in the yeast pichia pastoris for NMR studies.

Deuterium isotope labelling is important for NMR studies of large proteins and complexes. Many eukaryotic proteins are difficult to express in bacteria, but can be efficiently produced in the methylotrophic yeast Pichia pastoris. In order to facilitate NMR studies of the malaria parasite merozoite surface protein-1 (MSP1) complex and its interactions with antibodies, we have investigated production of the MSP1-19 protein in P. pastoris grown in deuterated media. The resulting deuteration patterns were analyzed by NMR and mass spectrometry. We have compared growth characteristics and levels of heterologous protein expression in cells adapted to growth in deuterated media (95% D2O), compared with expression in non-adapted cells. We have also compared the relative deuteration levels and the distribution pattern of residual protiation in protein from cells grown either in 95% D2O medium with protiated methanol as carbon source, or in 95% D2O medium containing deuterated methanol. A high level of uniform Calpha deuteration was demonstrated, and the consequent reduction of backbone amide signal linewidths in [1H/15N]-correlation experiments was measured. Residual protiation at different positions in various amino acid residues. including the distribution of methyl isotopomers, was also investigated. The deuteration procedures examined here should facilitate economical expression of 2H/13C/15N-labelled protein samples for NMR studies of the structure and interactions of large proteins and protein complexes.

NMR studies of ligand carboxylate group interactions with arginine residues in complexes of Lactobacillus casei dihydrofolate reductase with substrates and substrate analogues.

In a series of complexes of Lactobacillus casei dihydrofolate reductase (DHFR) formed with substrates and substrate analogues, the (1)H/(15)N NMR chemical shifts for the guanidino group of the conserved Arg 57 residue were found to be sensitive to the mode of binding of their H(eta) protons to the charged oxygen atoms in ligand carboxylate groups. In all cases, Arg 57 showed four nonequivalent H(eta) signals indicating hindered rotation about the N(epsilon)-C(zeta) and C(zeta)-N(eta) bonds. The H(eta)(12) and H(eta)(22) protons have large downfield shifts as expected for a symmetrical end-on interaction with the ligand carboxylate group. The chemical shifts are essentially the same in the complexes with folate and p-aminobenzoyl-L-glutamate (PABG) and similar to those found previously for the methotrexate complex reflecting the strong and similar hydrogen bonds formed with the carboxylate oxygens. Interestingly, the rates of rotation about the N(epsilon)-C(zeta) bond for the complexes containing the weakly binding PABG fragment are almost identical to those measured in the complex with methotrexate, which binds 10(7) times more tightly. In the methotrexate complex, this rotation depends on correlated rotations about the N(epsilon)-C(zeta) bond of Arg 57 and the C(alpha)-C' bond of the ligand glutamate alpha-carboxylate group. Thus, even in a fragment such as PABG, which has a much faster off-rate, the carboxylate group binds to the enzyme in a similar way to that in a parent molecule such as folate and methotrexate with the rotation about the N(epsilon)-C(zeta) bond of Arg 57 being essentially the same in all the different complexes.