Dominant and recessive aryl hydrocarbon hydroxylase-deficient mutants of mouse hepatoma line,Hepa-1, and assignment of recessive mutants to three complementation groups

Fifty-four benzo[a]pyrene (BP)-resistant, aryl hydrocarbon hydroxylase (AHH)-deficient mutants were found to be recessive, while five were dominant. Hybrids between the former mutants and the wild-type were killed by BP, and possessed AHH activities of at least 0.5 (relative to the wild-type). Dominant-mutant-wild-type hybrids were resistant to BP and had activities of about 0.05. Additional experiments assigned the recessive mutants to three complementation groups, designated A through C. Group-B-group-C hybrids were exceptional in possessing a mean AHH activity (0.36), less than the value (0.5) expected from gene dosage. This deficiency was probably due, in part, to instability of AHH activity in these hybrids. However, all hybrids tested retained stable DNA complements, equal to the sum of those of their parents, for 140 days in culture. Previous studies have shown that group B and group C mutations both affect the functioning of a cytosolic receptor required for AHH induction (1).     

Diurnal rhythms of leptin and ghrelin in the systemic circulation and in the gastric mucosa are related to food intake in rats

We investigated diurnal changes in leptin and ghrelin levels in the stomach and in the systemic circulation and their relation to food intake rhythms in Wistar rats housed at 22 °C with a 12-h light/dark cycle and free access to food and water. Animals were sacrificed every 3 h over a 24-h period. Leptin and ghrelin levels in serum and in the gastric mucosa were analysed by immunoassay. Leptin mRNA levels were determined in the gastric mucosa by RT-PCR and in different adipose tissue depots (epididymal, retroperitoneal and mesenteric) by Northern blot. Ghrelin mRNA levels were determined by Northern blot. Gastric and serum leptin levels displayed similar diurnal rhythms, rising during the dark phase and decreasing gradually during the light phase. Leptin expression in the different adipose tissue depots correlated positively with circulating leptin levels (P<0.05), although there were some depot-associated differences. Leptin mRNA levels in the mesenteric depot correlated positively with food intake (P<0.05). In blood, ghrelin levels rose sharply just before the onset of the dark phase and dropped suddenly just after. In the stomach, ghrelin levels were high during the fasting period of light and low during the night, and correlated inversely with food intake, gastric contents and serum leptin levels (P<0.05). Leptin and ghrelin in the stomach and in the systemic circulation thus show diurnal variations that are influenced by food intake rhythms. The results agree with a role for ghrelin as a stimulant of meal initiation.     

Neural responses to free field and virtual acoustic stimulation in the inferior colliculus of the guinea pig

Virtual auditory space (VAS) stimuli based on outer ear transfer functions became increasingly important in spatial hearing research. However, few studies have investigated the match between responses of auditory neurons to VAS and free-field (FF) stimulation. This study validates acoustic spatial receptive fields (SRFs) of 183 individual midbrain units using both VAS and FF stimuli. The first-spike latency, which varied systematically across SRFs, was 14.9 +/- 8.3 (SD) ms in FF, and 15.1 +/- 8.3 ms in VAS. Spike-count-based SRFs measured 0-20 dB above the neural threshold covered on average 44.5 +/- 18.0% of the recorded sphere in FF and 45.5 +/- 18.7% in VAS. The average deviation of the centroid position of SRFs using FF and VAS stimuli was 7.4 degrees azimuth and 3.3 degrees elevation. The average spike rate remained unchanged. The SRF overlap recorded using FF and VAS stimuli (mean: 71.3 +/- 12.6%) or repeated FF stimuli (70.2 +/- 14.2%) was high and strongly correlated (r = 0.96; P < 0.05). The SRF match observed with FF and VAS stimuli was not significantly altered over a range of stimulus levels (paired t-test P = 0.51; n = 6). Randomized VAS barely affected SRF sizes, centroids, or maximum spike count but decreased the average minimum response to 59% compared with sequential stimulation (paired t-test; P = 0.05; n = 26). SRF recordings in VAS excluding the acoustic distortions of the recording equipment differed from those in VAS incorporating the equipment (paired t-test P = 0.01; n = 5). In conclusion, neurophysiological recordings demonstrate that individualized VAS stimuli provided a good simulation of a FF environment.     

Second harmonic imaging of intrinsic signals in muscle fibers in situ

We use second harmonic generation (SHG) imaging to study and quantify a strong intrinsic SHG signal in skeletal muscle fiber preparations and single isolated myofibrils. The intrinsic signal follows the striation pattern of the muscle cells and is positioned at the sarcomeric location of the myosin filaments. Interestingly, the signal is enhanced at the region where the myosin heads are located on the myosin filaments. As the intrinsic signal reflects the subcellular structure in an accurate way, SHG can be used for noninvasive high resolution structural imaging without exogenous labels in living muscle cells. This may be very important for detecting changes in myofibrillar organization occurring under pathophysiological conditions, e.g., in cardiac and skeletal myopathies. Due to the strong dependency of SHG on orientation and symmetries of the tissue, it may allow the study of dynamic interactions between the contractile proteins actin and myosin during force production and muscle shortening. Furthermore, SHG imaging can be combined with other nonlinear microscopical techniques, such as laser scanning multiphoton fluorescence microscopy, to simultaneously measure other dynamic cellular processes, representing a complementary method and extending the range of nonlinear microscopical methods.     

Regulation of human basophil adhesion to endothelium under flow conditions: Different very late antigen 4 regulation on umbilical cord blood-derived and peripheral blood basophils

BACKGROUND: Although soluble mediators released by basophils in tissue sites contribute to the chronic injury that occurs in hypersensitivity diseases, only limited information is available about how circulating basophils are recruited to tissues. In particular, the interaction of basophils with endothelium under conditions that mimic physiologic flow has not been explored. OBJECTIVE: We sought to identify adhesion molecules regulating the attachment of human basophils to IL-4-activated human umbilical vein endothelial cells (HUVECs) under flow conditions. METHODS: A parallel-plate flow chamber and blocking mAbs were used to define the adhesion molecules involved in the interactions of peripheral blood basophils (PBBs) and cord blood-derived basophils (CBDBs) with IL-4-activated HUVECs and with Chinese hamster ovary (CHO) cell transfectants expressing specific adhesion molecules. A fluorescent ligand specific for very late antigen 4 (VLA-4) was used to directly examine the VLA-4 affinity state of basophils. RESULTS: Flowing PBBs and CBDBs attached to activated HUVECs and to CHO cells expressing P- or E-selectin. However, only CBDBs attached to vascular cell adhesion molecule 1 (VCAM-1)-transfected CHO cells under flow conditions. The attachment of CBDBs to CHO cells was blocked by mAbs directed against E-selectin, P-selectin, and VCAM-1, whereas attachment of PBBs was blocked by E-selectin and P-selectin mAbs. Activating VLA-4 with Mn(2+) on PBBs resulted in adhesion to the VCAM-1-transfected CHO cells, indicating that VLA-4 activity on PBBs can be regulated, at least in part, through affinity changes. The Mn(2+)-induced upregulation of basophil VLA-4 affinity was demonstrated directly by using a fluorescent ligand for VLA-4 and flow cytometry. CONCLUSIONS: The interaction of human CBDBs and PBBs with endothelium under flow conditions is mediated in part by both P- and E-selectin. VLA-4 additionally contributes to the adhesion of flowing CBDBs. However, the affinity of VLA-4 is too low to support the adhesion under flow conditions of unstimulated PBBs.     

Evaluation of hyperforin analogues for inhibition of 5-lipoxygenase

The acylphloroglucinol hyperforin, a constituent of the herb Hypericum perforatum (St. John's wort), was recently identified as potent and direct inhibitor of 5-lipoxygenase (5-LO), the key enzyme in the biosynthesis of proinflammatory leukotrienes. In this study, naturally occurring analogues of hyperforin, isolated from H. perforatum, as well as a series of synthetic derivatives obtained by chemical modification of hyperforin by acylation, alkylation or oxidation, were analysed for the inhibition of 5-LO. The efficacies of these compounds were evaluated in intact human polymorphonuclear leukocytes, but also the inhibitory effects on isolated recombinant human 5-LO were investigated. Our data show that some of the oxidised hyperforin derivatives possess even improved efficacy, whereas alkylation and acylation have detrimental effects.     

Homozygous microdeletion of chromosome 4q11-q12 causes severe limb-girdle muscular dystrophy type 2E with joint hyperlaxity and contractures

Microdeletion syndromes are commonly transmitted as dominant traits and are frequently associated with variably expressed pleiotropic phenotypes. Nonlethal homozygous microdeletions, on the other hand, are very rare. Here, we delineate the fifth and so far largest homozygous microdeletion in nonmalignancies of approximately 400 kb on chromosome 4q11-q12 in a large consanguineous East-Anatolian family with six affected patients. The deleted region contains the beta-sarcoglycan gene (SGCB), the predicted gene SPATA18 (spermatogenesis associated 18 homolog) and several expressed sequence tags. Patients presented with a severe and progressive Duchenne-like muscular dystrophy phenotype, a combination of hyperlaxity and joint contractures, chest pain, palpitations, and dyspnea.     

Phenotype delineation of ZNF462 related syndrome

Zinc finger protein 462 (ZNF462) is a relatively newly discovered vertebrate specific protein with known critical roles in embryonic development in animal models. Two case reports and a case series study have described the phenotype of 10 individuals with ZNF462 loss of function variants. Herein, we present 14 new individuals with loss of function variants to the previous studies to delineate the syndrome of loss of function in ZNF462. Collectively, these 24 individuals present with recurring phenotypes that define a multiple congenital anomaly syndrome. Most have some form of developmental delay (79%) and a minority has autism spectrum disorder (33%). Characteristic facial features include ptosis (83%), down slanting palpebral fissures (58%), exaggerated Cupid's bow/wide philtrum (54%), and arched eyebrows (50%). Metopic ridging or craniosynostosis was found in a third of study participants and feeding problems in half. Other phenotype characteristics include dysgenesis of the corpus callosum in 25% of individuals, hypotonia in half, and structural heart defects in 21%. Using facial analysis technology, a computer algorithm applying deep learning was able to accurately differentiate individuals with ZNF462 loss of function variants from individuals with Noonan syndrome and healthy controls. In summary, we describe a multiple congenital anomaly syndrome associated with haploinsufficiency of ZNF462 that has distinct clinical characteristics and facial features.