The Effect of Dietary Energy Restriction on Body Weight Gain and the Development of Noninsulin-Dependent Diabetes Mellitus (NIDDM) in Psammomys obesus

Food intake was restricted to 75% of ad libitum levels in 37 male Psammomys obesus (Israeli Sand Rats) from the ages of 4 (weaning) to 10 weeks. Energy restriction reduced the mean body weight at 10 weeks by 29% compared with 44 ad libitum fed controls. Hyperglycemia was prevented completely in the food-restricted group, and mean blood glucose concentrations were significantly reduced (3.8 ± 0.2 vs. 5.5 ± 0.4 μmol/L; p<0.05) compared with control animals. Plasma insulin concentrations were also decreased significantly compared with ad libitum fed controls (105 ± 13 vs. 241 ± 29 mU/L;p<0.05). Although energy restriction prevented hyperglycemia from developing in 10-week-old P. obesus, 19% of the food restricted animals still developed hyperinsu-linemia. We concluded that hyperphagia between the ages of 4 to 10 weeks may be essential for the development of noninsulin-dependent diabetes mellitus in P. obesus, but that hyperinsulinemia may still occur in the absence of hyperphagia and hyperglycemia, suggesting a significant genetic influence on the development of hyperinsulinemia in this animal model.     

Physical properties of oligonucleotides containing phosphoramidate-modified internucleoside linkages.

Because of their nuclease resistance and ability to form substrates for RNase H, antisense oligodeoxynucleotides (ODNs) possessing several methoxyethylphosphoramidate linkages at both termini have proven effective at targeting the degradation of specific mRNAs in Xenopus embryos. The efficacy of these compounds subsequently observed in tissue culture focused our attention on the issue of cellular uptake. To investigate the extent to which phosphate backbone modifications may increase the lipophilicity of ODNs, and thereby increase passive uptake by cells, the partitioning of a series of phosphoramidate-modified compounds between aqueous and organic phases was examined. The octanol:water partition coefficient of an unmodified, mixed-sequence 16-mer was 1.75 x 10(-5). The log of the partition coefficient increased in a sigmoidal manner with the number of methoxyethylphosphoramidate internucleoside linkages, indicating a nonlinear free energy relationship. The highest level of partitioning demonstrated was approximately 4 x 10(-3) (a 230-fold increase), attained when 11 of the 15 phosphodiesters were modified. An increase in hydrophobicity was also attained with C8 and C10 alkylamines acting as phase-transfer agents. The melting temperatures of heteroduplexes formed between a phosphoramidate-modified ODN and a complementary unmodified DNA strand decreased by approximately 1.5 degrees C for every phosphate group modification. ODNs can thus be extensively derivatized without substantially compromising duplex formation under physiological conditions.     

The interaction of hemoglobin with the cytoplasmic domain of band 3 of the human erythrocyte membrane

Previous studies point to the acidic amino-terminal segment of band 3, the anion transport protein of the red cell, as the common binding site for hemoglobin and several of the glycolytic enzymes to the erythrocyte membrane. We now report on the interaction of hemoglobin with the synthetic peptide AcM-E-E-L-Q-D-D-Y-E-D-E, corresponding to the first 11 residues of band 3, and with the entire 43,000-Da cytoplasmic domain of the protein. In the presence of increasing concentrations of the peptide, the oxygen binding curve for hemoglobin is shifted progressively to the right, indicating that the peptide binds preferentially to deoxyhemoglobin. The dissociation constant for the deoxyhemoglobin-peptide complex at pH 7.2 in the presence of 100 mM NaCl is 0.31 mM. X-ray crystallographic studies were carried out to determine the exact mode of binding of the peptide to deoxyhemoglobin. The difference electron density map of the deoxyhemoglobin-peptide complex at 5 A resolution showed that the binding site extends deep (approximately 18 A) into the central cavity between the beta chains, along the dyad symmetry axis, and includes Arg 104 beta 1 and Arg 104 beta 2 as well as most of the basic residues within the 2,3-diphosphoglycerate binding site. The peptide appears to have an extended conformation with only 5 to 7 of the 11 residues in contact with hemoglobin. In agreement with the crystallographic studies, binding of the peptide to deoxyhemoglobin was blocked by cross-linking the beta chains at the entrance to the central cavity. Oxygen equilibrium studies showed that the isolated cytoplasmic fragment of band 3 also binds preferentially to deoxyhemoglobin. The binding of the 43,000-Da fragment to hemoglobin was inhibited in the cross-linked derivative indicating that the acidic amino-terminal residues in the intact cytoplasmic domain also bind within the central cavity of the hemoglobin tetramer.     

Kinetic analysis of Escherichia coli RNase H using DNA-RNA-DNA/DNA substrates.

The kinetic properties of Escherichia coli ribonuclease H (RNase H) were investigated using oligonucleotide substrates that consist of a short stretch of RNA, flanked on either side by DNA (DNA-RNA-DNA). In the presence of a complementary DNA strand, RNase H cleavage is restricted to the short ribonucleotide stretch of the DNA/RNA heteroduplex. The DNA-RNA-DNA substrate utilized for kinetic studies: (formula; see text) is cleaved at a single site (decreases) in the presence of a complementary DNA strand, to generate (dT)7-(rA)2-OH and p-(rA)2-(dT)9. Anion exchange high performance liquid chromatography was used to separate and quantitate the cleavage products. Under these conditions, RNase H-specific and nonspecific degradation products could be resolved. Kinetic parameters were measured under conditions of 100% hybrid formation (1.2-1.5 molar excess of complementary DNA, T much less than Tm). A linear double reciprocal plot was obtained, yielding a Km of 4.2 microM and a turnover number of 7.1 cleavages per s per RNase H monomer. The kinetic properties of substrate analogs containing varying lengths of RNA (n = 3-5) and 2'-O-methyl modifications were also investigated. Maximal turnover was observed with DNA-RNA-DNA substrates containing a minimum of four RNA residues. Kcat for the rA3 derivative was decreased by more than 100-fold. The Km appeared to decrease with the size of the internal RNA stretch (n = 3-5). No significant difference in turnover number of Km was observed when the flanking DNA was replaced with 2'-O-methyl RNA, suggesting that RNase H does not interact with this region of the heteroduplex.     

Secondary structural changes in globular protein induced by a surfactant: Fourier self-deconvolution of FT-IR spectra

Conformational changes in ovalbumin, a globular protein, induced by an anionic surfactant, sodium dodecyl sulfate (SDS), have been monitored by an FT-IR spectrometer using ZnSe cylindrical internal reflection optics which allows high quality IR spectra to be obtained in water solution. The most notable change, on addition of SDS, occurs in the composite band of the Amide I absorption band and the vibrational frequency of the composite C = O bond shifts from 1639 cm-1 to 1652 cm-1. On the other hand, the position of the Amide II band remains fairly unchanged. Comparison of the various peak positions in the deconvoluted spectra for the native protein and the perturbed protein clearly shows the effect of SDS on the secondary structures of the protein. SDS unfolds the protein. It increases the helix content slightly. More importantly, it alerts the beta sheet structure, destroying it almost completely in the Amide I region, while retaining it in its neighbourhood. In the deconvoluted spectra of the perturbed protein, a band at 1531 cm-1 indicates generation of some beta turns. We used the second derivative of the deconvoluted spectra for fixing positions of minor peaks and shoulders. The results of this study indicate that the deconvolution of the normal IR spectra, consisting of composite bands, provides evidence for the specific secondary structures in a protein and for the way they are affected by changes in the environment, e.g., the addition of SDS. This makes it possible to relate conformational changes to specific secondary structures.     

The mutational dynamics of the SARS-CoV-2 virus in serial passages in vitro

Since its outbreak in 2019, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) keeps surprising the medical community by evolving diverse immune escape mutations in a rapid and effective manner. To gain deeper insight into mutation frequency and dynamics, we isolated ten ancestral strains of SARS-CoV-2 and performed consecutive serial incubation in ten replications in a suitable and common cell line and subsequently analysed them using RT-qPCR and whole genome sequencing. Along those lines we hoped to gain fundamental insights into the evolutionary capacity of SARS-CoV-2 in vitro. Our results identified a series of adaptive genetic changes, ranging from unique convergent substitutional mutations and hitherto undescribed insertions. The region coding for spike proved to be a mutational hotspot, evolving a number of mutational changes including the already known substitutions at positions S:484 and S:501. We discussed the evolution of all specific adaptations as well as possible reasons for the seemingly inhomogeneous potential of SARS-CoV-2 in the adaptation to cell culture. The combination of serial passage in vitro with whole genome sequencing uncovers the immense mutational potential of some SARS-CoV-2 strains. The observed genetic changes of SARS-CoV-2 in vitro could not be explained solely by selectively neutral mutations but possibly resulted from the action of directional selection accumulating favourable genetic changes in the evolving variants, along the path of increasing potency of the strain. Competition among a high number of quasi-species in the SARS-CoV-2 in vitro population gene pool may reinforce directional selection and boost the speed of evolutionary change.     

Beacon interacts with cdc2/cdc28-like kinases

Previously we found elevated beacon gene expression in the hypothalamus of obese Psammomys obesus. Beacon administration into the lateral ventricle of P. obesus stimulated food intake and body weight gain. In the current study we used yeast two-hybrid technology to screen for proteins in the human brain that interact with beacon. CLK4, an isoform of cdc2/cdc28-like kinase family of proteins, was identified as a strong interacting partner for beacon. Using active recombinant proteins and a surface plasmon resonance based detection technique, we demonstrated that the three members of this subfamily of kinases (CLK1, 2, and 4) all interact with beacon. Based on the known sequence and functional properties of beacon and CLKs, we speculate that beacon could either modulate the function of key regulatory molecules such as PTP1B or control the expression patterns of specific genes involved in the central regulation of energy metabolism.     

Effect of γ radiation on physiological parameters of the ethanolic fermentation

This work examines the efficacy of radiation in reducing the viability of certain contaminating bacteria of sugar-cane must and the consequential beneficial effect of lethal doses of radiation on some physiological parameters of the yeast-based ethanolic fermentation. The must from sugar-cane juice was inoculated with different bacteria that usually contaminate the must in the production facilities: Bacillus and Lactobacillus. The contaminated must was irradiated at 2.0, 4.0, 6.0, 8.0 and 10.0 kGy of gamma radiation. The population density of the bacteria in the irradiated must was recorded. Ethanolic fermentation by yeast (Saccharomyces cerevisiae) was carried out and the total acidity, the volatile acidity and the organic acids (lactic and acetic) produced during the fermentation were determined. The ethanol yield was also recorded. The treatment with radiation reduced the population of the contaminating microorganisms of the sugar-cane must. The acidity and the organic acids (lactic and acetic) produced during the fermentation decreased as the dose of radiation applied to the must increased. It is concluded that irradiation was efficient in decontaminating the sugar-cane must and improved the biochemical parameters of the ethanolic fermentation, including the ethanol yield by 2%.     

Linkage of infantile Bartter syndrome with sensorineural deafness to chromosome 1p.

Bartter syndrome (BS) is a family of disorders manifested by hypokalemic hypochloremic metabolic alkalosis with normotensive hyperreninemic hyperaldosteronism. We evaluated a unique, inbred Bedouin kindred in which sensorineural deafness (SND) cosegregates with an infantile variant of the BS phenotype. Using a DNA-pooling strategy, we screened the human genome and successfully demonstrated linkage of this unique syndrome to chromosome 1p31. The genes for two kidney-specific chloride channels and a sodium/hydrogen antiporter, located near this region, were excluded as candidate genes. Although the search for the disease-causing gene in this family continues, this linkage further demonstrates the genetic heterogeneity of BS. In addition, the cosegregation of these phenotypes allows us to postulate that a single genetic alteration may be responsible for the SND and the BS phenotype. The identification and characterization of this gene would lead to a better understanding of the normal physiology of the kidney and the inner ear.     

Pulmonary atresia with ventricular septal defect: a case for central venous pressure and oxygen saturation monitoring.

A 21-year-old patient with pulmonary atresia and ventricular septal defect (PA-VSD) was admitted to the hospital for tubal ligation. Invasive arterial and central venous (CVP) pressure, pulse oximetric oxygen saturation (SpO2), and (from the tip of oximetric central venous catheter) central venous oxygen saturation (ScvO2) and oxygen extraction rate (ExO2) were continuously monitored. Heart rate (range: 68-75 beat/min), mean arterial pressure (80-90 mmHg), CVP (7-10 mmHg), SpO2 (79-90 percent), ScvO2 (57-70 percent), and ExO2 (21-30 percent) remained stable during epidural anesthesia and transvaginal sterilization. Following an overnight stay (peak SpO2 92 percent; peak ScvO2 71 percent; through ExO2 21 percent), the oxygen data returned to baseline on awakening (SpO2 < 80 percent, ScvO2 < 55 percent, ExO2 > 35 percent), and the patient was discharged. In PA-VSD, a single-outlet double-ventricle anomaly, CVP reflects the preload of systemic ventricle. As the mixed venous oxygen saturation cannot be defined, ScvO2 is the best available indicator of the whole body oxygen consumption. Continuous monitoring of CVP, ScvO2 and ExO2 in the superior vena cava may provide more insight into the response to anesthesia and surgery in patients with PA-VSD.