Genetic and molecular analysis of familial isolated growth hormone deficiency

Familial isolated growth hormone deficiency (IGHD) has been associated with complete deletions of the hGH-N gene encoding the pituitary growth hormone (GH) in a large number of cases. However, there is still no alternative empirical explanation for the remaining familial or non-familial IGHD cases. We studied a large kindred including five IGHD-affected first cousins to determine possible IGHD inheritance and whether the hGH-N gene was the cause of IGHD in this pedigree. Sex-linked and autosomal recessive transmission of IGHD in this kindred was rejected. Autosomal dominant inheritance was the most probable explanation according to a model of one locus with two alleles, one being dominant for IGHD, under genetic modifiers or epistasis. Southern blotting analysis (BamHI and HindIII digestions) was used to determine whether the hGH-N gene was present in the patients and their family members. Because we found that the hGH-N gene was present, five restriction fragment length polymorphisms (RFLPs; HincII, MspI-A and B, and BglII-A and B) linked to the hGH-N gene were used to try to identify the possible RFLP haplotypes in the pedigree that could be markers or associated with the abnormal hGH-N alleles responsible for IGHD. From the haplotype analysis, it appeared that other genes not linked to the hGH-N gene cluster were the cause of the IGHD phenotype in this kindred. An alternative conclusion could be that the hGH-N gene was responsible for IGHD in this kindred, if a mutation (gene conversion) at the MspI-B site or a reciprocal recombination event between the HincII and MspI-B sites occurred from generation P to F1 and a similar event took place from generation F1 to F2. The non-significant GH responses of patients to the growth releasing factor test confirmed that the hGH-N gene structural product or some step in its regulation was responsible for causing IGHD in this kindred. We suggest that genetic micromutations in the hGH-N gene are present and are responsible for IGHD. We developed a method using the polymerase chain reaction to amplify a 790-bp fragment of the hGH-N gene. The fragment spanned from the second part of the dyad symmetry region in the non-transcribed 5 end of the hGH-N gene to 9 bp before the alternative splice-acceptor site in exon 3. The expected fragment was verified by its digestion with seven diagnostic resctriction endonucleases (BamHI, FspI, PstI, NdeI, BssHII, BglII and HincII). The results showed no deletions or insertions greater than 35 bp in the hGH-N amplified fragment from the DNAs of the IGHD patients and their family members.Presented, in part, at the VIth International Congress of Auxology, Madrid, Spain, 15–19 September 1991.     

Purification and chemical modifications of porphobilinogen oxygenase

Porphobilinogen oxygenase from wheat germ was purified and was found to be a cationic protein containing 8 mol of nonheme iron and 8–10 mol of labile sulfide per mole of enzyme (M_r, 100,000). The enzyme isolated from either wheat germ or rat liver microsomes was found to exist in multiple molecular weight forms. When succinylated, only one molecular weight form of 25,000 was obtained and it retained full activity. It had lost all of the sigmoidal kinetics characteristic of the native enzyme. While the native enzyme had an n = 3.5, the succinylated enzyme showed Michaelian kinetics. A K_m of approximately 1.70 mm was determined for the succinylated wheat germ enzyme, and a K_m of approximately 2.5 mm was found for the succinylated microsomal enzyme. Acetylation of the enzyme afforded an active acetylated enzyme which showed allosteric kinetics and multiple molecular weight forms. The products formed by the succinylated enzyme were the same as those formed by the native enzyme.     

Solution structure and stability against digestion of rproBnIb, a recombinant 2S albumin from rapeseed: relationship to its allergenic properties

NMR spectroscopy has been used to determine the solution structure of the precursor form of the recombinant napin BnIb, rproBnIb, a 2S albumin, 109-residue protein from the seeds of Brassica napus. More than 90% of the side-chain proton resonances were unambiguously assigned from the analysis of two-dimensional correlation (COSY), total correlation (TOCSY), and nuclear Overhauser effect (NOESY) spectra. The final structures were computed by using restrained molecular dynamics on the basis of 1316 upper-limit distance constraints derived from NOE cross-correlation intensities. The computed structures exhibited a root-mean-square deviation (RMSD) radius of 0.66 A for the backbone and 1.16 A for the side-chain heavy atoms of the structural core. The resulting structure consists of five amphipathic helices arranged in a right-handed super helix, a folding motif found in other proteins of the prolamin superfamily. As in the case of the mature protein, the recombinant precursor behaves as a plant food allergen. To trace out the origin and characteristics of its allergenic properties, rproBnIb was assayed against simulated gastric fluid and found to be very resistant to proteolysis. Also, heat treatment of the protein followed up to 85 degrees C by circular dichroism showed a very limited unfolding, which was recovered after cooling to 20 degrees C, indicating a high thermal stability. These results suggest that rproBnIb, as other 2S albumins, may be able to reach the gut immune system intact. A comparison of the putative epitopes against IgE antibodies of the three members of the prolamine family [2S albumins, nonspecific lipid transfer proteins (nsLTPs), and alpha-amylase/trypsin inhibitors] indicates that there are not common surfaces of interaction with IgE. Though the epitopes appear to be located in different regions of the proteins, they do comply with the requirements of being solvent-exposed and flexible.     

A major allergen from pollen defines a novel family of plant proteins and shows intra- and interspecies [correction of interspecie] cross-reactivity

Olive tree (Olea europaea) pollen is a main cause of allergy associated with extensive areas of Europe and North America. Ole e 10, a small (10.8 kDa) and acidic (pI 5.8) protein, has been identified as a major allergen from the olive pollen, isolated, and characterized. Circular dichroism analysis gave 17% alpha helix, 33% beta sheet, and 21% beta turn for its secondary structure. Based on amino acid sequences of tryptic peptides, the protein was cloned and sequenced. The allergen consists of a single polypeptide chain of 102 aa, with a signal peptide of 21 residues. Ole e 10 showed homology with the C-terminal domain of another olive allergen, Ole e 9 (1,3-beta-glucanase, 53% identity), with deduced sequences from Arabidopsis thaliana genes (42-46% identity) and with polypeptide segments (Cys boxes) of proteins involved in yeast development (Epd1/Gas-1p/Phr2 families; 42-43% similarity). Ole e 10 showed 55% prevalence for olive-allergic patients and exhibited an IgE response dependent on its conformation. Remarkable IgE cross-reactivity was detected with Ole e 9, but no correlation was observed between the individual IgE responses to both allergens. Ole e 10 shares IgE B cell epitopes with proteins from Oleaceae, Gramineae, Betulaceae, Chenopodiaceae, Cupressaceae, Ambrosia, and Parietaria pollens, latex, and vegetable foods, such as tomato, kiwi, potato, and peach. These data indicate that Ole e 10 is a new pan-allergenic plant protein that shows notable intra- and interspecie IgE cross-reactivity and is a powerful candidate to be involved in pollen-latex-fruit syndrome.     

Amlodipine decreases fibrosis and cardiac hypertrophy in spontaneously hypertensive rats: persistent effects after withdrawal

Our objective was to examine the effect of chronic treatment with amlodipine on blood pressure, left ventricular hypertrophy, and fibrosis in spontaneously hypertensive rats and the persistence of such an effect after drug withdrawal. We investigated the effects of treatment with 2, 8 and 20 mg/kg/day of amlodipine given orally for six months and at three months after drug withdrawal. Systolic blood pressure was measured using the tail-cuff method. At the end of the study period, the heart was excised, the left ventricle was isolated, and the left ventricle weight/body weight ratio was calculated as a left ventricular hypertrophy index. Fibrosis, expressed as collagen volume fraction, was evaluated using an automated image-analysis system on sections stained with Sirius red. Age-matched untreated Wistar-Kyoto and SHR were used as normotensive and hypertensive controls, respectively. Systolic blood pressure was reduced in the treated SHR in a dose-dependent way and after amlodipine withdrawal it increased progressively, without reaching the values of the hypertensive controls. Cardiac hypertrophy was reduced by 8 and 20 mg/kg/day amlodipine, but when treatment was withdrawn only the group treated with 8 mg/kg/day maintained significant differences versus the hypertensive controls. All three doses of amlodipine reduced cardiac fibrosis and this regression persisted with the two highest doses after three months without treatment. We concluded that antihypertensive treatment with amlodipine is accompanied by a reduction in left ventricular hypertrophy and regression in collagen deposition. Treatment was more effective in preventing fibrosis than in preventing ventricular hypertrophy after drug withdrawal.     

In vivo autofluorescence in the biological windows: the role of pigmentation

Small animal deep‐tissue fluorescence imaging in the second Biological Window (II‐BW, 1000–1350 nm) is limited by the presence of undesirable infrared‐excited, infrared‐emitted (900–1700 nm) autofluorescence whose origin, spectral properties and dependence on strains is still unknown. In this work, the infrared autofluorescence and laser‐induced whole body heating of five different mouse strains with distinct coat colors (black, grey, agouti, white and nude) has been systematically investigated. While neither the spectral properties nor the magnitude of organ autofluorescence vary significantly between mouse strains, the coat color has been found to strongly determine both the autofluorescence intensity as well as the laser‐induced whole body heating. Results included in this work reveal mouse strain as a critical parameter that has to be seriously considered in the design and performance of small animal imaging experiments based on infrared‐emitting fluorescent markers.

     

Modular diversification of the locomotor system in damselfishes (Pomacentridae)

As fish move and interact with their aquatic environment by swimming, small morphological variations of the locomotor system can have profound implications on fitness. Damselfishes (Pomacentridae) have inhabited coral reef ecosystems for more than 50 million years. As such, habitat preferences and behavior could significantly constrain the morphology and evolvability of the locomotor system. To test this hypothesis, we used phylogenetic comparative methods on morphometric, ecological and behavioral data. While body elongation represented the primary source of variation in the locomotor system of damselfishes, results also showed a diverse suite of morphological combinations between extreme morphologies. Results show clear associations between behavior, habitat preferences, and morphology, suggesting ecological constraints on shape diversification of the locomotor system. In addition, results indicate that the three modules of the locomotor system are weakly correlated, resulting in versatile and independent characters. These results suggest that Pomacentridae is shape may result from the interaction between (1) integrated parts of morphological variation that maintain overall swimming ability and (2) relatively independent parts of the morphology that facilitate adaptation and diversification. J. Morphol. 277:603–614, 2016. © 2016 Wiley Periodicals, Inc.     

Bite Force in Four Pinniped Species from the West Coast of Baja California,Mexico, in Relation to Diet,Feeding Strategy,and Niche Differentiation

Behavioral foraging differences are known to aid in food resource partitioning in pinniped communities, but it is not known whether skull biomechanical efficiency also contributes to dietary niche partitioning. We tested this hypothesis in a community of four sympatric species of pinnipeds that co-occur along the coast of Baja California: California sea lion (Zalophus californianus), northern elephant seal (Mirounga angustirostris), harbor seal (Phoca vitulina), and Guadalupe fur seal (Arctocephalus townsendi). We tested whether their preferred prey items differed in resistivity to puncture and whether those differences were linked to the mass of the muscles of mastication and the biomechanical efficiency with which they can puncture prey items. For each prey species, we measure resistivity to puncture using texture profile analysis. We found that M. angustirostris consumes the most resistant prey and that A. townsendi consumes the least resistant. We estimated physiological cross-sectional area of the muscles of mastication for each pinniped and found that the same pair of species respectively has the largest and smallest theoretical value of muscular force. Finally, we estimated the bite force that each pinniped species requires to puncture its prey by solving Euler-Lagrange equations based on biomechanical lever model parameters measured from 3D digital models of the skulls. We also found differences in efficiency between the species. These data allowed us to classify the three ecomorphological types. Type 1 features a hydrodynamic skull with relatively low mandibular forces, characteristic of pelagic carnivore feeders such as A. townsendi. Type 2, represented by Z. californianus and M. angustirostris (both opportunistic feeders), is characterized by broad insertion areas for the mandibular muscles and strong teeth, permitting these predators to vary the prey target species as a function of prey availability. Type 3 features a less robust skull and a lower muscle efficiency, characteristic of benthic feeders such as P. vitulina. This evidence indicates that biomechanical differences between the species contribute to dietary niche construction.