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We report on a four-generation inbred family including 10 individuals affected with a form of craniotubular dysplasia (CTD). All affected patients were born to consanguineous healthy parents; this finding, together with the equal sex ratio among affected individuals and the occurrence of only normal individuals among their offspring, indicates that the disease in this family is an autosomal recessive (AR) trait. Taking into account the segregation pattern of the disease in the family and the radiological characteristics of two young CTD patients, the most likely diagnosis for the defect is AR craniometaphyseal dysplasia (CMD). CMD is a CTD, with both autosomal dominant (AD) and recessive forms. The description of the present genealogy confirms the AR pattern of inheritance of some cases of CMD and contributes to a better delineation of the clinical spectrum of AR CMD, suggesting a more pronounced diaphyseal involvement in the AR compared with the AD CMD. Through genomewide scanning, we mapped the AR CMD to a 7 cM interval, between D6S302 and D6S1639, at 6q21-22 region. We have also excluded the positional candidate COL10A1 gene as being the responsible for this disorder. Curiously, a form of AR spondylocostal dysplasia (SD) also segregates in the family, including one affected individual with both conditions. The gene DLL3, mapped to 19q13 region, was recently found to be responsible for one form of AR SD; however, we did not find evidence of linkage between this 19q region and the SD segregating in our family, thus implying in genetic heterogeneity for AR SD., (Copyright 2000 Wiley-Liss, Inc.)

Ehlers-Danlos syndrome (EDS) type VIIC is a recessively inherited connective-tissue disorder, characterized by extreme skin fragility, characteristic facies, joint laxity, droopy skin, umbilical hernia, and blue sclera. Like the animal model dermatosparaxis, EDS type VIIC results from the absence of activity of procollagen I N-proteinase (pNPI), the enzyme that excises the N-propeptide of type I and type II procollagens. The pNPI enzyme is a metalloproteinase containing properdin repeats and a cysteine-rich domain with similarities to the disintegrin domain of reprolysins. We used bovine cDNA to isolate human pNPI. The human enzyme exists in two forms: a long version similar to the bovine enzyme and a short version that contains the Zn++-binding catalytic site but lacks the entire C-terminal domain in which the properdin repeats are located. We have identified the mutations that cause EDS type VIIC in the six known affected human individuals and also in one strain of dermatosparactic calf. Five of the individuals with EDS type VIIC were homozygous for a C-->T transition that results in a premature termination codon, Q225X. Four of these five patients were homozygous at three downstream polymorphic sites. The sixth patient was homozygous for a different transition that results in a premature termination codon, W795X. In the dermatosparactic calf, the mutation is a 17-bp deletion that changes the reading frame of the message. These data provide direct evidence that EDS type VIIC and dermatosparaxis result from mutations in the pNPI gene.

The tetramerization of melittin, a 26-amino acid peptide from Apis mellifera bee venom, has been studied as a model for protein folding. Melittin converts from a monomeric random coil to an alpha-helical tetramer as the pH is raised from 4.0 to 9.5, as ionic strength is increased, as temperature is raised or lowered from about 37 degrees C, or as phosphate is added. The thermodynamics of this tetramerization (termed "folding") are explored using circular dichroism. The melittin tetramer has two pKa values of 7.5 and 8.5 corresponding to protonation of the N-terminus and Lys 23, respectively. pKa values calculated with the program DelPhi (Gilson, M.K., Sharp, K.A., & Honig, B.H., 1987, J. Comp. Chem. 9, 327-335; Gilson, M.K. & Honig, B.H., 1988a, Proteins 3, 32-52; Gilson, M.K. & Honig, B.H., 1988b, Proteins 4, 7-18) agree with experimental titration data. Greater electrostatic repulsion of these protonated groups destabilizes the tetramer by 3.6 kcal/mol at pH 4.0 compared to pH 9.5. Increasing the concentration of NaCl in the solution from 0 to 0.5 M stabilizes the tetramer by 5-6 kcal/mol at pH 4.0. The effect of NaCl is modeled with a ligand-binding approach. The melittin tetramer is found to have a temperature of maximum stability ranging from 35.5 to 43 degrees C depending on the pH, unfolding above and below that temperature. delta Cp0 for folding ranges from -0.085 to -0.102 cal g-1 K-1, comparable to that of other small globular proteins (Privalov, P.L., 1979, Adv. Protein Chem. 33, 167-241). delta H0 and delta S0 are found to decrease with temperature, presumably due to the hydrophobic effect (Kauzmann, W., 1959, Adv. Protein Chem. 14, 1-63). Phosphate is found to perturb the equilibrium substantially with a maximal effect at 150 mM, stabilizing the tetramer at pH 7.4 and 25 degrees C by 4.6 kcal/mol. The enthalpy change due to addition of phosphate (-7.5 kcal/mol at 25 degrees C) can be accounted for by simple dielectric screening. Both circular dichroism and crystallographic results suggest that phosphate may bind Lys 23 at the ends of the elongated tetramer. These detailed measurements give insight into the relative importance of various forces for the stability of melittin in the folded form and may provide an experimental standard for future tests of computational energetics on this simple protein system.

The present report describes our experience with 16 adolescents and young adults with insulin-dependent diabetes mellitus (IDDM; Type I) who switched from two injections of insulin per day to the NovolinPen and four insulin injections a day. Their mean age at the initiation of therapy was 19.2 years with a duration of diabetes of 8.9 years. Sixteen random computer-matched (for age, sex, and duration of diabetes) controls who remained on two insulin injections a day were evaluated for comparison. Most subjects were preparing to leave home for college. After a minimum of 1 year of using the pen, the mean weight gain of the test subjects was significantly greater (P less than .05) than for the computer-matched controls. Glycemic control, as monitored by mean HbA1c values, were similar for the NovolinPen and the control groups (10.8 versus 10.9, respectively) after almost 2 years. The main advantage to the pen expressed by the users was a sense of freedom of lifestyle.

Flow velocity waveforms (FVWs) from the fetal umbilical artery were recorded from 2178 pregnant women over a 6-year period. All of them had an obstetric factor indicating increased risk of fetal compromise. A total of 6749 studies was recorded. The systolic diastolic (AB) ratio was measured and classified as normal (less than 95th centile), elevated (95-99th centile), high (greater than 99th centile) or extreme (absent diastolic flow). The results of these studies have been related to subsequent fetal and neonatal outcome. An abnormal umbilical artery FVW was associated with shorter gestation and infants with lower birthweight, shorter length and lower ponderal index. There was a highly significant association between an abnormal FVW and the birth of an infant small for gestational age. The significance of the association increased with the increased abnormality of the umbilical artery FVW and this was independent of gestational age. Preterm infants associated with high or extreme AB ratios spent twice as long in the neonatal nursery than those with normal AB ratios. Analysis of 794 pregnancies studies serially indicated that an abnormal FVW in which the AB ratio was increasing, in contrast to a decreasing AB ratio, predicted a poor outcome for both size at birth and duration of neonatal intensive care. We conclude that in high risk pregnancy Doppler umbilical artery FVW studies predict the most compromised fetuses in terms of growth retardation and requirements for neonatal intensive care.

The importance of the hydrophobic interaction in stabilizing native protein structure has long been appreciated. However, more than other component forces, this one has resisted quantitative description. We present two approximate methods of assessing the hydrophobic component to the free energy of protein folding. Both are expressed in terms of what can be called hydrophobic moments of the protein. The first method is intended to yield an approximate value for the hydrophobic energy. This energy is calculated from a set of atomic coordinates in terms of the hydrophobicity (or 0th hydrophobic moment) of each amino acid residue and its accessibility or lack of it to aqueous solvent. The second method considers the first moment of the hydrophobicity of a group of residues, the hydrophobic moment. Segments of secondary structure in folded proteins tend to have hydrophobic moments that oppose each other. For example, alpha-helices on the protein surface tend to have one hydrophobic face and one hydrophilic face, with the hydrophilic face out towards the solvent. This pattern of organization is often apparent from a computer model of the protein that shows the magnitude and direction of the hydrophobic moment of each segment of secondary structure. Examples are given for the incorrectly folded structures of Novotný et al [J Mol Biol 177:787, 1984] and for the correct structures to which they correspond.

Twelve- and sixteen-residue peptides have been designed to form tetrameric alpha-helical bundles. Both peptides are capable of folding into amphiphilic alpha-helices, with leucyl residues along one face and glutamyl and lysyl residues along the opposite face. Four such amphiphilic alpha-helices are capable of forming a noncovalently bonded tetramer. Neighboring helices run in antiparallel directions in the design, so that the complex has 222 symmetry. In the designed tetramer, the leucyl side chains interdigitate in the center in a hydrophobic interaction, and charged side chains are exposed to the solvent. The designed 12-mer (ALPHA-1) has been synthesized, and it forms helical aggregates in aqueous solution as judged by circular dichroic spectroscopy. It has also been crystallized and characterized by x-ray diffraction. The crystal symmetry is compatible with (but does not prove) the design. The design can be extended to a four-alpha-helical bundle formed from a single polypeptide by adding three peptide linkers.

The specific binding of tritiated ouabain was used to estimate the density of Na+-K+-ATPase sites ("Na+-pump" sites) in segments of skeletal muscle from normal and dystrophic mice. Ouabain binding was approximately 4 times greater in red (soleus) muscle than in white (superficial gastrocnemius) muscle from normal animals. In dystrophic soleus muscles, ouabain binding was decreased by nearly one-half. Because Na+-K+-ATPase activity is associated with plasma membranes, these observations constitute further evidence for a sarcolemmal abnormality in dystrophic mice.