A new hereditary single band variant of the Gc system

Summary A new single band variant (Gc Ar) or the Gc subtypes not identical with the known Gc variants has been detected in the plasma of a healthy blood donor by isoelectric focusing. Using this technique the variant is represented by a single band which has a similar isoelectric point to the Gc 1C2 anodal band. It is well known that the single band Gc phenotypes remain unaltered after neuraminidase treatment. Nevertheless, the new single band variant (Gc Ar) is altered after neuraminidase treatment as is Gc 2A3. After neuraminidase treatment, the Gc Ar band is affected and moved to the nearby position of the Gc 2 band. Investigation of the proband's family shows that the variant occurs combined with the common alleles Gc 1F, Gc 1S and that it has an autosomal dominant inheritance.     

Fermentation Studies with Streptomyces griseus: II. Synthetic Media for the Production of Streptomycin

Synthetic media for streptomycin fermentation were studied to determine which media gave highest yields of streptomycin. The effect of salts on streptomycin production by Streptomyces griseus was examined, and a suitable combination of salts was established in a glucose-casein medium. This medium yielded 3,000 μg/ml of the antibiotic with an inoculum of 1.6%. Substitution of amino acids for casein was examined. Of 17 amino acids tested, best results were obtaind with sodium aspartate. Substitution of ammonium salts was tried, and an excellent streptomycin yield was obtained with a medium containing ammonium citrate.     

Immunocytochemical localization of 5α-reductase type 1 in the prostate of normal and castrated rats

We immunohistochemically studied the localization of 5-reductase type 1 in combination with androgen receptor (AR) expression in individual lobes of the prostates of intact and castrated rats. In the normal rat prostate, 5-reductase was localized in the cytoplasm of most epithelial cells in the ventral, dorsal, and lateral type 1 (L1) lobes. Epithelial cells of lateral type 2 (L2) lobes were negative for 5-reductase. AR was present in the nuclei of all epithelial and stromal cells throughout the prostate. The number of 5-reductase-immunoreactive cells rapidly decreased in the ventral and L1 lobes after castration, whereas many positive cells remained in the dorsal lobe even at 4 weeks after castration. AR immunostaining was lost in the ventral, dorsal, and L1 lobes at 1 week after castration, but remained in the L2 lobe of 4-week-castrated rats. Electron microscopic immunocytochemistry showed that 5-reductase was exclusively localized in the rough endoplasmic reticulum membranes and that there were no distinct structural differences between the positively and negatively stained epithelial cells. These findings suggested that the expression of 5-reductase type 1 in the epithelial cell is heterogeneous within and among the individual lobes of the rat prostate, and does not correspond to AR expression.     

Anatomy of pharynx in patients with obstructive sleep apnea and in normal subjects

Isono, Shiroh, John E Remmers, Atsuko Tanaka, Yasuhide Sho,Jiro Sato, and Takashi Nishino. Anatomy of pharynx in patients with obstructive sleep apnea and in normal subjects.J. Appl. Physiol. 82(4):1319-1326, 1997.Anatomic abnormalities of the pharynx arethought to play a role in the pathogenesis of obstructive sleep apnea(OSA), but their contribution has never been conclusively proven. Thepresent study tested this anatomic hypothesis by comparing themechanics of the paralyzed pharynx in OSA patients and in normalsubjects. According to evaluation of sleep-disordered breathing (SDB)by nocturnal oximetry, subjects were divided into three groups: normalgroup (n = 17), SDB-1(n = 18), and SDB-2(n = 22). The static pressure-arearelationship of the passive pharynx was quantified under generalanesthesia with complete paralysis. Age and body mass index werematched among the three groups. The site of the primary closure was thevelopharynx in 49 subjects and the oropharynx in only 8 subjects.Distribution of the location of the primary closure did not differamong the groups. Closing pressure(PC) of the velopharynx forSDB-1 and SDB-2 groups (0.90 ± 1.34 and 2.78 ± 2.78 cmH₂O, respectively) wassignificantly higher than that for the normal group (3.77 ± 3.44 cmH₂O;P < 0.01). Maximal velopharyngealarea for the normal group (2.10 ± 0.85 cm²) was significantly greaterthan for SDB-1 and SDB-2 groups (1.15 ± 0.46 and 1.06 ± 0.75 cm², respectively). Theshape of the pressure-area curve for the velopharynx differed betweennormal subjects and patients with SDB, being steeper in slope nearPC in patients with SDB.Multivariate analysis of mechanical parameters and oxygen desaturationindex (ODI) revealed that velopharyngealPC was the only variable highly correlated with ODI. VelopharyngealPC was associated withoropharyngeal PC, suggestingmechanical interdependence of these segments. We conclude that thepassive pharynx is more narrow and collapsible in sleep-apneic patientsthan in matched controls and that velopharyngeal PC is the principal correlate ofthe frequency of nocturnal desaturations.

     

Interaction of cross-sectional area, driving pressure, and airflow of passive velopharynx

Isono, Shiroh, Thom R. Feroah, Eric A. Hajduk, Rollin Brant,William A. Whitelaw, and John E. Remmers. Interaction ofcross-sectional area, driving pressure, and airflow of passive velopharynx. J. Appl. Physiol. 83(3):851-859, 1997.Previous studies have shown that, when thepharyngeal muscles are relaxed, the velopharynx is a highly compliantsegment of the pharynx. Thus, under these circumstances,cross-sectional area of the velopharynx (A_VP), drivingpressure across the velopharynx (P), and inspiratory airflow(I) willbe mutually interdependent variables. The purpose of the presentinvestigation was to describe the interrelation among these threevariables during inspiration. We studied 15 sleeping patients withobstructive sleep apnea/hypopnea when the pharyngeal muscles wererendered hypotonic by applying continuous positive airway pressure tothe nasal airway.A_VP, determined by endoscopic imaging, was significantly greater at onset ofI limitationthan at minimum oropharyngeal pressure(P < 0.01). Snoring was neverobserved duringIlimitation. In a subgroup of six patients, values for P,I, andA_VP were obtainedat 0.1-s intervals at various levels of mask pressure. For these sixpatients, the mathematical expressionI = 0.657(A_VP/A_max) · P^0.332,where A_max ismaximal A_VP,described the relationship among the three variables(R² = 0.962) forflow-limited and non-flow-limited inspirations. The impedance of thepassive velopharynx, defined asP^0.33/,was inversely related toA_VP and increaseddramatically when A_VP was <0.3cm². In summary, we observed aprogressive decrease inA_VP during flow-limited inspiration in patients with obstructive sleep apnea. Thisconstriction of the velopharynx contributes to an increase invelopharyngeal impedance that, in turn, counterbalances the increase inP during flow limitation.

     

Transgenic Fluorescent Plasmodium cynomolgi Liver Stages Enable Live Imaging and Purification of Malaria Hypnozoite-Forms

A major challenge for strategies to combat the human malaria parasite Plasmodium vivax is the presence of hypnozoites in the liver. These dormant forms can cause renewed clinical disease after reactivation through unknown mechanisms. The closely related non-human primate malaria P. cynomolgi is a frequently used model for studying hypnozoite-induced relapses. Here we report the generation of the first transgenic P. cynomolgi parasites that stably express fluorescent markers in liver stages by transfection with novel DNA-constructs containing a P. cynomolgi centromere. Analysis of fluorescent liver stages in culture identified, in addition to developing liver-schizonts, uninucleate persisting parasites that were atovaquone resistant but primaquine sensitive, features associated with hypnozoites. We demonstrate that these hypnozoite-forms could be isolated by fluorescence-activated cell sorting. The fluorescently-tagged parasites in combination with FACS-purification open new avenues for a wide range of studies for analysing hypnozoite biology and reactivation.     

Dock8 interacts with Nck1 in mediating Schwann cell precursor migration

During embryonic development of the peripheral nervous system (PNS), Schwann cell precursors migrate along neuronal axons to their final destinations, where they will myelinate the axons after birth. While the intercellular signals controlling Schwann cell precursor migration are well studied, the intracellular signals controlling Schwann cell precursor migration remain elusive. Here, using a rat primary cell culture system, we show that Dock8, an atypical Dock180-related guanine-nucleotide exchange factor (GEF) for small GTPases of the Rho family, specifically interacts with Nck1, an adaptor protein composed only of Src homology (SH) domains, to promote Schwann cell precursor migration induced by platelet-derived growth factor (PDGF). Knockdown of Dock8 or Nck1 with its respective siRNA markedly decreases PDGF-induced cell migration, as well as Rho GTPase activation, in precursors. Dock8, through its unique N-terminal proline-rich motif, interacts with the SH3 domain of Nck1, but not with other adaptor proteins composed only of SH domains, e.g. Grb2 and CrkII, and not with the adaptor protein Elmo1. Reintroduction of the proline-rich motif mutant of Dock8 in Dock8 siRNA-transfected Schwann cell precursors fails to restore their migratory abilities, whereas that of wild-type Dock8 does restore these abilities. These results suggest that Nck1 interaction with Dock8 mediates PDGF-induced Schwann cell precursor migration, demonstrating not only that Nck1 and Dock8 are previously unanticipated intracellular signaling molecules involved in the regulation of Schwann cell precursor migration but also that Dock8 is among the genetically-conservative common interaction subset of Dock family proteins consisting only of SH domain adaptor proteins.     

A tightly regulated expression system for <Emphasis Type="Italic">E. coli</Emphasis> using supersaturated silicic acid

Objective

To develop a new expression system regulated by a ferric uptake regulator in which silicic acid is used as an inducer.

Results

Fur box (binding site for Fur) was substituted for the lac operator to regulate the expression of GFP with the lac promoter. Since the addition of supersaturated silicic acid invokes iron deficiency, supersaturated silicic acids were used as an inducer. GFP expression was dependent on silica concentration, and the expression level without silica was negligible. Basal expression level of this lac-Fur system was extremely low and, hence, lytic enzyme gene E from bacteriophage ?X174 could be retained in this system. Furthermore, the expression of genes of interest was spontaneously initiated as the cell density increased and the costs of the inducer are considerably less than IPTG.

Conclusion

The combination of lac promoter and Ferric uptake repressor allowed the protein expression by supersaturated silicic acid as an inducer in an easy and cost-effective way.