Duane T. Brandau

Probing protein structure and dynamics by second-derivative ultraviolet absorption analysis of cation–π interactions

We describe an alternate approach for studying protein structure using the detection of ultraviolet (UV) absorbance peak shifts of aromatic amino acid side chains induced by the presence of salts. The method is based on the hypothesis that salt cations (Li+, Na+, and Cs+) of varying sizes can differentially diffuse through protein matrices and interact with benzyl, phenyl, and indole groups through cation–π interactions. We have investigated the potential of this method to probe protein dynamics by measuring high resolution second‐derivative UV spectra as a function of salt concentration for eight proteins of varying physical and chemical properties and the N‐acetylated C‐ethyl esterified amino acids to represent totally exposed side chains. We show that small shifts in the wavelength maxima for Phe, Tyr, and Trp in the presence of high salt concentrations can be reliably measured and that the magnitude and direction of the peak shifts are influenced by several factors, including protein size, charge, and the local environment and solvent accessibility of the aromatic groups. Evaluating the empirical UV spectral data in light of known protein structural information shows that probing cation–π interactions in proteins reveals unique information about the influence of structure on aromatic side chain spectroscopic behavior.

Near-infrared photoacoustic spectroscopy of proteins

A major problem encountered with the use of electronic spectroscopy in the analysis of biological materials in the ultraviolet, visible, and infrared region involves the limited range of the physical state of samples that can be examined. In an attempt to expand this range, photoacoustic spectra of both solid- and solution-state proteins have been obtained in the near-infrared region. Solid proteins generate detailed spectra in the region 1.0-2.6 micron, resulting primarily from hydrogenic overtone and combinational modes. Harmonics and combinations of amide group frequencies which display significant spectral complexity are observed between 1.4 and 1.7 micron, although they appear to manifest only limited conformational sensitivity. Solution spectra in D2O are of much lower resolution. Assignments of peaks for both solution- and solid-state proteins are presented and the advantages and disadvantages of the use of near-infrared photoacoustic spectroscopy with proteins are discussed.

Autistic and dysmorphic features associated with a submicroscopic 2q33.3–q34 interstitial deletion detected by array comparative genomic hybridization†

Recently, Bisgaard et al. [2006] detected additional chromosomal abnormalities in six patients with previously known abnormal karyotypes, such as inversions and translocations, using metaphase comparative genomic hybridization (CGH) at a resolution of 2–3 Mb. They reported previously undetected interstitial deletions with CGH in their patients including a de novo 2q33.2–q34 deletion. However, no chromosome imbalances were found using conventional cytogenetics in their six patients at the inversion or translocation breakpoints. Deletions in the long arm of chromosome 2 are relatively rare and correlate with a broad spectrum of clinical findings, including developmental delay, mental retardation, hyperactivity with autistic traits and dysmorphic features including Pierre Robin sequence, cleft palate, temporal bone abnormalities and hypoplastic lungs [Kramer et al., 2000; Houdayer et al., 2001; Van Buggenhout et al., 2005; Pescucci et al., 2007]. Approximately one-half of the reported deletions involved a chromosome segment between bands 2q24.3 and 2q31.1 [Courtens et al., 1997] or terminal 2q37.3 deletions [Kitsiou-Tzeli et al., 2007]. In 1997, Courtens et al. reported the first 2q33.3– q34 interstitial deletion in a 5-week-old boy with phenotypic findings initially suggesting Seckel syndrome (e.g., preand postnatal growth retardation, microcephaly, and muscular hypotonia). Courtens et al. [1997] summarized seven cases involving at least the 2q33.3–q34 chromosome region (i.e., largest deletion 2q33–q36). Subsequently, four other cases have been reportedwith deletions in the 2q33– q35 region [Kramer et al., 2000; Riegel et al., 2001; Pescucci et al., 2003; Bisgaard et al., 2006]. Herein, we report a second case of a de novo 2q33.3–q34deletion in a 6-year-oldmale ascertained with aCGH and referred for evaluation of a de novo balanced translocation [t(4;16)(q32.2;q24.1)]. We compared findings in our case to those reported by Courtens et al. [1997], Pescucci et al. [2003], and Bisgaard et al. [2006] involving similar 2q deletions. Our patient was born to a para 4, gravid 4, 27-yearold mother and a 43-year-old father. The mother denied alcohol or drug exposure during pregnancy. The infant was delivered at term by an uncomplicated vaginal delivery with a birth weight of 3,182 g (25th centile) and length of 50.8 cm (50th centile). The head circumference was not known. The infant was discharged with the mother at 24 hr of age. During the first year of life, the patient was hospitalized on three occasions for respiratory infections (RSV and pneumonia). Sweat chloride testing for cystic fibrosis was negative. Asthma and severe allergies were diagnosed requiring treatment with loratadine, montelukast sodium, and a budesonide inhaler. Tympanostomy tubes were placed at 6 months and again at 18 months of age for persistent otitis media. The child had extensive dental caries requiring restoration at 3.5 years of age. Developmental milestones were delayed (e.g., sitting alone at 1 year, first steps at 18–24 months and first words at 24 months). At 2 years of age the

Kinetics of the precipitation of cryoimmunoglobulins

The kinetics of the cryoprecipitation of two monoclonal IgG and two monoclonal IgM cryoimmunoglobulins, two IgM/IgG mixed cryoglobulins and a series of cold soluble monoclonal IgG and IgM immunoglobulins in the presence of polyethylene glycol have been compared by time dependent turbidity measurements. The effects of temp and ionic strength on kinetic processes are described in detail. The monoclonal cryoimmunoglobulins display lag times which are not seen with the other proteins, suggesting a critical nucleation event. The protein concn dependence of the lag times indicate that these nucleation centers contain only a few immunoglobulin molecules. Direct evidence for the existence of precipitation nuclei was obtained from dynamic light scattering studies of two of the monoclonal proteins during their lag periods. Both proteins manifested an approx. 20% decrease in their mean diffusion coefficients (corresponding to a 25% increase in Stokes radius) prior to detectable precipitation. This suggests the formation of nuclei between 2 and 8 times the size of the monomeric proteins. It is postulated that the increasing size of mixed cryoglobulin complexes with decreasing temp provides analogous nucleation sites. The latter stages of precipitation appear to be kinetically similar for all proteins examined, although the size and shape of the aggregates are quite variable.

Follicle stimulating and leutinizing hormones, estradiol and testosterone in Prader–Willi syndrome†

Prader–Willi syndrome (PWS) is a classic genomic imprinting disorder in which affected individuals display hypotonia, failure to thrive, feeding difficulties, developmental delays and hypogenitalism/ hypogonadism in infancy.At approximately2–3years of age, hyperphagia, central obesity, short stature, small hands and feet, behavioral problems and characteristic facial features are present [Cassidy, 1984; Butler, 1990; Bittel and Butler, 2005; Butler et al., 2006]. The genetic causes are generally due to a paternal deletion of the chromosome 15q11–q13 region (about 70%), maternal disomy 15 (UPD) (about 25%), or imprinting defects or translocations involving chromosome 15 (about 5%) [Bittel and Butler, 2005]. The characteristic findings of hyperphagia, central obesity, short stature, cryptorchidism and hypogonadism are consistent with endocrine and/or metabolic abnormalities involving the hypothalamic–pituitary axis. While some studies have examined specific hormonal levels in Prader–Willi syndrome [Nagai et al., 1998; Burman et al., 2001; Eiholzer and Lee, 2006; Eiholzer et al., 2006; Butler et al., 2007], a comprehensive examination of follicle stimulating hormone (FSH), leutinizing hormone (LH), estradiol and testosterone levels is lacking in individuals with Prader–Willi syndrome with known genetic subtypes. Thus, we report FSH, LH, estradiol and testosterone levels in a relatively large cohort of males and females (16 years of age and older) with Prader–Willi syndrome as a function of genetic subtype and obesity status. The regulation of FSH and LH secretion and subsequent estradiol (female) and testosterone (male) secretion is highly integrated involving multiple signaling pathways, receptor systems and signaling molecules such as inhibin and ghrelin [Ojeda et al., 2006]. In females, FSH levels vary throughout the menstrual cycle peaking at mid-cycle while LH levels peak prior to ovulation. Both FSH and LH levels increase dramatically after menopause. Hypothalamic hypogonadism subjects are characterized by lowFSH, LHand sexhormone levelswhile normal or elevated FSH levels indicate a gonadal origin. Elevated LH levels are seen in females with ovarian failure while low levels are seen in pituitary failure. Inmales, there is a prominent neonatal surge of LH, FSH and testosterone that begins shortly after birth and peaks at 6–8 weeks of age before gradually waning over 4–6 months [Veldhuis et al., 2006]. This early rise in FSH and LH is not observed in females. At 10 years of age for females and at 11 years of age in males, adrenarche usually occurs as defined by the appearance of pubic and axillary hair and increased apocrine sweat gland activity. This event is triggered by increased adrenal productionof dehydroepiandrosterone, dehydroepiandrosterone sulfate and androstenedione but not cortisol [Veldhuis et al., 2006]. However, maturation of the testes and ovaries (gonadarche) requires the orderly activation of the hypothalamic–pituitary–gonadal axis. The initiation of this process remains poorly defined but involves increased pulsatile secretion of gonadotropin releasing hormone (GnRH) that subsequently controls FSH and LH secretion from the anterior pituitary [Veldhuis et al., 2006]. This orderly

Lack of binding between cryoimmunoglobulins, immunoglobulins and fibronectin: implications for immune complex vasculitis

Immunc‐complex‐mcdiated vasculitis is a frequent complication of rheumatoid arthritis and systemic lupus erythematosus. The mechanism of deposition of immune complexes within the vessel wall in these diseases remains unknown, but probably involves other proteins. Fibronectin is a likely candidate since it possesses the ability to bind to collagen, endothelial cells, and possibly immunoglobulins and immune complexes. In this study, the hinding of libronectin to IgG and IgM cryoglobulins, cold soluble IgM, IgG, IgG subclasses and IgG fragments was investigated in the solution phase. Static light scattering, fluorescence anisotropy. fluorescence intensity, and PEG precipitation studies were used to investigate binding under different conditions of temperature and ionic strength. These studies failed to demonstrate significant binding between fibronectin and IgM, IgG, IgG subclasses and IgG fragments under the conditions studied These findings argue against solution phase binding of fibronectin and immunoglobulins contributing to immune complex vasculitis. The possibility of important surface interactions between these proteins has not been ruled out.

Cortisol levels in Prader–Willi syndrome support changes in routine care†

The authors would like to draw the reader’s attention to a change in authorship in the following article: Invited Comment—Cortisol Levels in Prader–Willi Syndrome Support Changes in Routine Care. Am J Med Genet A 149A:138–139. The names of two contributors who were initially involved in this work, Duane T. Brandau, DO, Ph.D. (now at the Rocky Vista University College of Osteopathic Medicine) and Mariana Theodoro, BS were not included. The authors regret this oversight. The list of authors should therefore read: Merlin G. Butler, Duane T. Brandau, Mariana Theodoro, and Uttam Garg.

The interaction of cryoimmunoglobulins with a model surface

Cryoimmunoglobulins are associated with numerous clinical problems ranging from collagen vascular disorders (rheumatoid arthritis and systemic lupus erythematosus) to infectious processes including HIV infection. The precise role of cryoglobulins in the pathophysiology of these disorders remains unresolved. Although cold insolubility may account for some of the observed processes, it cannot explain the entire array of findings in cryoglobulinemia. An alternative hypothesis suggests that the subtle differences responsible for cold precipitation of these proteins renders them intrinsically more sticky, resulting in deposition of cryoimmunoglobulins on vascular surfaces. We have explored this hypothesis by characterizing the binding of monoclonal cold soluble and cryoimmunoglobulins to silica beads as a model biological surface. It is found that monoclonal, type I, IgM and IgG cryoglobulins have only a slight tendency to bind to a greater extent to this surface than cold soluble immunoglobulins. Physical studies utilizing front surface fluorescence measurements and differential scanning calorimetry show surface interaction leads to partial thermal destabilization of the proteins. To a limited extent, this destabilization is more pronounced with the cryoglobulins compared to cold-soluble control homologues. Surface bound IgM cryoimmunoglobulin was also found to fix complement less efficiently than their cold soluble surface bound counterparts. These studies do not strongly support the hypothesis that pathological mechanisms of cryoimmunoglobulins primarily involve abnormal surface interactions, although surface effects could play a limited role in some situations.

Morning melatonin levels in Prader–Willi syndrome†

Prader–Willi syndrome (PWS) is a classical genomic imprinting disorder characterized by hypotonia, hypogonadism, hyperphagia, central obesity, short stature, small hands and feet and learning/ behavioral problems due to a paternal deletion of chromosome 15q11–q13 region (about 70% of cases), maternal disomy 15 (UPD) (about 25%), or imprinting defects or translocations involving chromosome 15 (about 5%) [Butler, 1990; Bittel and Butler, 2005; Cassidy and Driscoll, 2009]. Several of these characteristics are consistent with endocrine and/or metabolic abnormalities involving the hypothalamic-pituitary axis. Similarly, findings of daytime drowsiness, obesity, sleep apnea and possibly scoliosis which are frequently seen in PWS may be linked to melatonin production and function [Bartness et al., 2002; Azeddine et al., 2007]. Two earlier studies reported normal melatonin levels in PWS subjects; however, the studies were limited by sample size, children only, and lack of genetic subtype information [Tamarkin et al., 1982; Willig et al., 1986]. Melatonin is secreted by the pineal gland in synchronization to light/dark cycles achieving maximum plasma concentrations of approximately 200 pg/ml at night and lower daytime levels of approximately 10 pg/ml [Cavallo, 1992; de Leersnyder et al., 2001]. Melatonin is produced from tryptophan with serotonin as an intermediate compound while high dietary tryptophan can elevate plasma levels. It can affect the autonomic regulation of the cardiovascular system, regulation of the hypothalamic– pituitary–gonadal axis, circadian rhythms and wake-sleep cycles [Cavallo, 1993; Commentz and Helmke, 1995; Nishiyama et al., 2001]. Melatonin levels are linked to energy expenditure and body mass regulation [Bartness et al., 2002] with obese pubertal males having an overall increase in melatonin production and release [Fideleff et al., 2006]. Because several PWS features could be influenced by melatonin, morning plasma melatonin levels were measured in 53 PWS subjects (25 males and 28 females) with a mean age of 23.6 years (range 10–50 years). All blood samples were drawn in the clinic setting under standard room lighting conditions. About 60% of the blood samples were collected between 7 and 8 AM and the remaining subjects between 8 and 11 AM with an average time of 8 AM. Ninety percent of the subjects were Caucasian. Age, obesity status, gender, genetic subtype, history of daytime drowsiness, sleep apnea, scoliosis, bone density measures, and diabetes status were recorded as well as any use of selective serotonin reuptake inhibitors (SSRIs), insulin and/or growth hormone. All subjects had genetic testing including DNA methylation, cytogenetic analysis with FISH and/or genotyping of informative markers from the 15q11–q13 region to identify the typical deletion type (type I or type II) or maternal disomy (UPD) as previously described [Bittel and Butler, 2005]. No PWS subjects were on thyroid or growth hormone, but four males were on testosterone and 12 were currently on SSRIs. Ten subjects were diabetic and four were on insulin. No PWS individuals were taking melatonin or dietary supplements with tryptophan. Body mass index (BMI) was calculated (kg/m) for each subject and obesity defined as BMI>30 for adults (>18 years) and a BMI>95% using published standards for sex for subjects less than 18 years [Kuczmarski et al., 2000]. Demographic data and melatonin levels are summarized in Table I. Daytime drowsiness and sleep apnea were determined by the use of questionnaire forms and history at the time of enrollment and sleep apnea verified by reviewing medical records including sleep assessment studies. Bone density measurements were determined by dual X-ray absorptiometry (DEXA) scans at the time of blood collection and scoliosis determined by physical examination and inspection of DEXA scans as previously described [Butler et al., 2001]. Fasting plasma morning melatonin levels were determined by a double-antibody radioimmunoassay (RIA) using a melatonin direct RIA kit purchased from ALPCO Diagnostics (Salem, NH)

The Effect of Interchain Disulfide Bond Cleavage on the Cold Induced Precipitation of Cryoimmunoglobulins

Selective cleavage of the interchain disulfide bonds present in the two IgG1-kappa monoclonal cryoglobulins Ger and Muk results in a partial loss of cryoprecipitability of the parent proteins at 0 degree C. The progressive loss of cryoprecipitability which occurs as a function of increasing reductant concentration parallels the successive cleavage of interheavy-light and interheavy-heavy chain disulfides. Circular dichroism shows that reduction and alkylation of hinge region disulfides induces small conformational changes in the IgG molecules that could alter cryoprecipitability. The N-terminal amino acid sequence of the Fc component derived by restricted proteolysis with trypsin of protein Muk was found to be completely homologous with N-terminal Fc sequences of noncryoglobulin IgG reference proteins, indicating identical hinge regions. Reduction and alkylation of two monoclonal IgM cryoglobulins also reduces cryoprecipitability. After reduction and alkylation of either the monoclonal IgM rheumatoid factor or the polyclonal IgG component of two mixed cryoglobulins recombination results in decreased cryoprecipitation of the intact cryoglobulin complex. In all cases inhibition of cryoprecipitation is greater when iodoacetic acid rather than iodoacetamide is employed as the S-alkylating group. These results do not support a direct role for the hinge region in the precipitation of cryoimmunoglobulins.