The enthalpy of protolysis of liver alcohol dehydrogenase upon binding nicotinamide adenine dinucleotide.

The binding of NAD+, NADH, and ADP-ribose to horse liver alcohol dehydrogenase has been studied calorimetrically as a function of pH at 25 degrees C. The enthalpy of NADH binding is 0 +/- 0.5 kcal mol-1 in the pH range 6 to 8.6. The enthalpy of NAD+ binding, however, varies with pH in a sigmoidal fashion and is -4.0 kcal mol(NAD)-1 at pH 6.0 and +4.5 kcal mol(NAD)-1 at pH 8.6 with an apparent pKa of 7.6 +/- 0.2. The enthalpy of proton ionization of the group on the enzyme is calculated to be in the range 8.8 to 9.8 kcal mol(H+)-1. In conjunction with the available thermodynamic data on the ionization of zinc-bound water in model compounds, it is concluded that the group with a pKa of 9.8 in the free enzyme and 7.6 in the enzyme . NAD+ binary complex is, most likely, the zinc-bound water molecule. Our studies with zinc-free enzyme provide further evidence for this conclusion. Therefore, the processes involving a conformational change of the enzyme upon NAD+ binding and the suggested mechanism of subsequent quenching of the fluorescence of Trp-314 implicating the participation of an ionized tyrosine group must be re-evaluated in the light of this thermodynamic study.     

Mapping and ordering of fragments of BK virus DNA produced by restriction endonucleases.

A total of 51 restriction sites were recognized within the BK virus genome by the combination of 10 different restriction endonucleases. These sites were mapped and oriented relative to one another as well as to the five fragments generated by the digestion of BK virus DNA with HindIII and EcoRI. The result was a comprehensive physical map suitable for in-depth characterization of the functions of BK virus at the molecular level.     

Ribosomal protein L35: identification in spinach chloroplasts and isolation of a cDNA clone encoding its cytoplasmic precursor

We describe the isolation of spinach chloroplast ribosomal protein L35 and characterization of a cDNA clone encoding its cytoplasmic precursor. This protein was only recently identified in ribosomes, but the sequences of four L35 genes have now been reported and confirm its presence in eubacteria, chloroplasts, and cyanelles. Using N-terminal sequence data, oligonucleotides were designed and a cDNA library was screened. The nucleotide sequence of the cDNA clones shows that the spinach L35 protein is encoded as a precursor of 159 residues, comprising a mature protein of 73 residues and a transit peptide of 86 residues. The cleavage site for forming the mature protein is deduced to be Thr-Val-Phe-Ala decreases Ala-Lys-Gly-Tyr. The L35 protein in the photosynthetic organelle of the protozoan Cyanophora paradoxa is encoded in the organelle DNA [Bryant & Stirewalt (1990) FEBS Lett. 259, 273-280]. The corresponding gene has not been found in the chloroplast DNA of a lower plant (liverwort) and two higher plants. Our results demonstrate that the L35 protein in a higher plant (spinach) is encoded in the nucleus. This finding, in light of the endosymbiont hypothesis, suggests an organelle to nucleus transfer of the L35 gene at the evolutionary beginnings of land plants.     

Structure of guanylyl-3',5'-cytidine monophosphate. II. Description of the molecular and crystal structure of the calcium derivative in space group P2(1).

The structural features of calcium guanosine-3′,5′-cytidine monophosphate (GpC) have been elucidated by X-ray diffraction analysis. The molecule was crystallized in space group P2₁ with cell constants of a = 21.224 Å, b = 34.207 Å, c = 9.327 Å, and β = 90.527°, Z = 8. The hydration of the crystal is 21% by weight with 72 water molecules in the unit cell. The four GpC molecules in the asymmetric unit occur as two Watson-Crick hydrogen-bonded dimers related by a pseudo-C face centering. Each dimer consists of two independent GpC molecules whose bases are hydrogen bonded to each other in the traditional Watson-Crick fashion. Each dimer possesses a pseudo twofold axis broken by a calcium ion and associated solvent. The four molecules are conformationally similar to helical RNA, but are not identical to it or to each other. Instead, values of conformational angles reflect the intrinsic flexibility of the molecule within the range of basic helical conformations. All eight bases are anti, sugars are all C3′-endo, and the C4′-C5′ bond rotations are gauche-gauche. The R factor is 12.6% for 2918 observed reflections at 1.2-Å resolution.     

Integration of a multicomponent intervention for hypertension into primary healthcare services in Singapore—A cluster randomized controlled trial

BackgroundDespite availability of clinical practice guidelines for hypertension management, blood pressure (BP) control remains sub-optimal (<30%) even in high-income countries. This study aims to assess the effectiveness of a potentially scalable multicomponent intervention integrated into primary care system compared to usual care on BP control.Methods and findingsA cluster-randomized controlled trial was conducted in 8 government clinics in Singapore. The trial enrolled 916 patients aged ≥40 years with uncontrolled hypertension (systolic BP (SBP) ≥140 mmHg or diastolic BP (DBP) ≥90 mmHg).Multicomponent intervention consisted of physician training in risk-based treatment of hypertension, subsidized losartan-HCTZ single-pill combination (SPC) medications, nurse training in motivational conversations (MCs), and telephone follow-ups. Usual care (controls) comprised of routine care in the clinics, no MC or telephone follow-ups, and no subsidy on SPCs. The primary outcome was mean SBP at 24 months’ post-baseline. Four clinics (447 patients) were randomized to intervention and 4 (469) to usual care. Patient enrolment commenced in January 2017, and follow-up was during December 2018 to September 2020. Analysis used intention-to-treat principles. The primary outcome was SBP at 24 months. BP at baseline, 12 and 24 months was modeled at the patient level in a likelihood-based, linear mixed model repeated measures analysis with treatment group, follow-up, treatment group × follow-up interaction as fixed effects, and random cluster (clinic) effects.A total of 766 (83.6%) patients completed 2-year follow-up. A total of 63 (14.1%) and 87 (18.6%) patients in intervention and in usual care, respectively, were lost to follow-up. At 24 months, the adjusted mean SBP was significantly lower in the intervention group compared to usual care (−3.3 mmHg; 95% CI: −6.34, −0.32; p = 0.03). The intervention led to higher BP control (odds ratio 1.51; 95% CI: 1.10, 2.09; p = 0.01), lower odds of high (>20%) 10-year cardiovascular risk score (OR 0.67; 95% CI: 0.47, 0.97; p = 0.03), and lower mean log albuminuria (−0.22; 95% CI: −0.41, −0.02; p = 0.03). Mean DBP, mortality rates, and serious adverse events including hospitalizations were not different between groups. The main limitation was no masking in the trial.ConclusionsA multicomponent intervention consisting of physicians trained in risk-based treatment, subsidized SPC medications, nurse-delivered motivational conversation, and telephone follow-ups improved BP control and lowered cardiovascular risk. Wide-scale implementation of a multicomponent intervention such as the one in our trial is likely to reduce hypertension-related morbidity and mortality globally.Trial registrationTrial Registration: Clinicaltrials.gov {"type":"clinical-trial","attrs":{"text":"NCT02972619","term_id":"NCT02972619"}}NCT02972619.

Tazeen H Jafar and colleagues present findings from a cluster-randomized controlled trial conducted to evaluate the effectiveness of an intervention designed to manage hypertension.     

The amino and carboxyl termini of perilipin a facilitate the storage of triacylglycerols

Perilipin A is the most abundant lipid droplet-associated protein in adipocytes and serves important functions in regulating triacylglycerol levels by reducing rates of basal lipolysis and facilitating hormonally stimulated lipolysis. We have previously shown that the central region of perilipin A targets and anchors it to lipid droplets, at least in part via three moderately hydrophobic sequences that embed the protein into the hydrophobic core of the droplet. The current study examines the roles of the amino and carboxyl termini of perilipin A in facilitating triacylglycerol storage. Amino- and carboxyl-terminal truncation mutations of mouse perilipin A were stably expressed in 3T3-L1 preadipocytes, which lack perilipins. Triacylglycerol content of the cells was quantified as a measure of perilipin function and was compared with that of cells expressing full-length perilipin A or control cells lacking perilipins. The amino-terminal sequence between amino acids 122 and 222, including four 10-11-amino acid sequences predicted to form amphipathic beta-strands and a consensus site for cAMP-dependent protein kinase, and the carboxyl terminus of 112 amino acids that is unique to perilipin A were critical to facilitate triacylglycerol storage. The precocious expression of full-length perilipin A in 3T3-L1 preadipocytes aided more rapid storage of triacylglycerol during adipose differentiation. By contrast, the expression of highly truncated amino- or carboxyl-terminal mutations of perilipin failed to serve a dominant negative function in lowering triacylglycerol storage during adipose differentiation. We conclude that the amino and carboxyl termini are critical to the function of perilipin A in facilitating triacylglycerol storage.