Identification of functioning sweat pores and visualization of skin temperature patterns in X-linked hypohidrotic ectodermal dysplasia by whole body thermography

Summary In this preliminary study, non-invasive infrared thermography has been used to visualize individual sweat pores and whole body skin temperature patterns in subjects with X-linked hypohidrotic ectodermal dysplasia (XHED) and normal controls. The findings in eight obligate heterozygotes and four affected males were compared to six normal female controls and to six non-manifesting females at risk for carrier status. Sweat secretion from individual pores in circumscribed areas was imaged using a high spatial resolution SPRITE infrared detector system working in the 8–14 m band. In seven out of eight obligate heterozygotes, skin areas devoid of active sweat glands were found on the face, the hands or the trunk. Tear front movement over the cornea was also visualized and abnormal patterns were identified in obligate heterozygotes. Whole body skin temperature patterns, obtained with an Agema 780 Medical Thermovision system, identified abnormal skin temperature distributions, including characteristic aberrant cas-cade back patterns, in obligate carriers. Two out of six at risk females had skin temperature patterns comparable with obligate heterozygotes and we have tentatively concluded that they are carriers. Thermal imaging may be used for the examination of at risk non-manifesting females in families with a single affected male. The results of this study suggest that the random X-inactivation in females with XHED, as well as producing relatively large skin areas with sweat pore aplasia, is also associated with abnormal temperature patterns that are consistent with altered peripheral vascular perfusion.     

Steroid-induced regulation of 3α,20β- and 3β,17β-hydroxysteroid dehydrogenase activity in wild type and mutants of Streptomyces hydrogenans

The effect of estradiol, hydrocortisone and progesterone on 3,20-and 3,17-hydroxysteroid dehydrogenase (HSD) in mutants of Streptomyces hydrogenans was compared to the steroid response of the wild type. Mutants were defective in arginine biosynthesis and/or aerial mycelial formation and lacked both enzymes or only 17-HSD. Some 17-HSD mutants had lost the ability to be induced by estradiol, by progesterone or by both. Some 20-HSD mutants had lost the ability to be induced by hydrocortisone, by progesterone or by both. Non-inducibility of 17-and 20-HSD by progesterone was not co-ordinate. An additional study of the growth phase-dependent enzyme activity of the wild type after induction with estradiol, hydrocortisone and progesterone was performed.Non-standard abbreviations 17-HSD 3,17-Hydroxysteroid dehydrogenase (EC 1.1.1.51) - 20-HSD 3,20-hydroxysteroid dehydrogenase (EC 1.1.1.53) - AO acridine orange - EBr ethidium bromide - EMS ethyl methanesulfonate - MNNG N-methyl-N-nitro-N-nitrosoguanidine     

Polyphosphoinositide mono- an diphosphoesterases of three subfractions of rat brain myelin

Phosphomonoesterase and diesterase that cleave phosphatidylinositol-4-phosphate (diphosphoinositide, DPI) and phosphatidylinositol-4,5-bisphosphate (triphosphoinositide, TPI) were detected in three subfractions of purified rat brain myelin, and some properties of the enzymes were studied. Monoesterase activity was stimulated by KCl, maximally at a concentration of 25 mM, and inhibited at KCl concentrations above 50 mM. Addition of boiled pH 5 supernatant of rat brain homogenate doubled the enzymic activity; EDTA was inhibitory. The specific activities were nearly equal in the low density, medium density, and heavy density myelin fractions but about 30% lower than in whole brain homogenate. The monophosphatase could be solubilized by extraction with 0.2% Triton X-100. The phosphodiesterase activity was inhibited by EDTA and EGTA and not stimulated by KCl or pH 5 supernatant. Specific activities were nearly equal in whole brain and myelin but were by about 60 percent elevated in the heavy density over the low density myelin fraction. These results show that the hydrolases operative in the fast turnover of the inositide phosphate groups are distributed over the entire myelin structure.     

Enzymatic syntheses of GlcNAcβ1-2Man and Galβ1-4GlcNAcβ1-2Man as components of complex type sugar chains

GlcNAc1-2Man and GlcNAc1-6Man were synthesized using the reverse hydrolysis activity of -N-acetylglucosaminidase from both jack beans and Bacillus circulans. In turn, Gal1-4GlcNAc1-2Man and Gal1-4GlcNAc1-6Man were synthesized regioselectively using the transglycosylation activity of -galactosidase from Diplococcus pneumoniae and B. circulans, respectively. These di- and trisaccharides are important components of complex type sugar chains and will be used as intermediates in our synthetic studies. Abbreviations: pNp--GlcNAc, p-nitrophenyl 2-acetamido-2-deoxy--D-glucopyranoside; pNp--Gal, p-nitrophenyl -D-galacto-pyranoside     

Differential expression of 2′∶3′-cyclic nucleotide 3′-phosphodiesterase in cultured central,peripheral, and extraneural cells

The relative levels of the central nervous system myelin marker enzyme 2:3-cyclic nucleotide 3-phosphodiesterase (EC 3.1.4.37, CNPase) were determined in neuroblastoma, astrocyte, oligodendrocyte and Schwann cell cultures and in freshly isolated human lymphocytes and platelets. The highest specific activities were associated with the cells that elaborate myelin membrane in the central and peripheral nervous system, oligodendrocytes and Schwann cells, respectively. Antiserum to bovine CNPase recognized both CNP1 and CNP2 in CNS myelin and human oligodendroglioma. In addition, a 53,000 dalton protein was evident on autoradiographs of immunoblotted PNS myelin and human oligodendroglioma proteins. Cultured rat oligodendrocyte, C₆ and mouse NA neuroblastoma CNPase appear to share common determinants with the corresponding normal rat CNS enzyme.     

Lectin cytochemical evaluation of somatosensory neurons and their peripheral and central processes in rat and man

Summary Paraffin sections of the trigeminal nerve root of the rat, and human spinal nerve root and trigeminal ganglion were stained with a battery of lectin-horseradish peroxidase conjugates to localize and characterize glycoconjugate (GC) in situ. In the rat the myelin sheath of the peripheral segment contained GC with sialic acid most probably linked to the penultinate disaccharide galactose(1 4)-N-acetylglucosamine (Gal(1 )-GlcNAc), and complex type N-glycosidic side chains. The myelin sheath in the central segment differed in containing little if any of the GC named above and in containing GC with terminal -Gal linked to N-acetylgalactosamine (GalNAc), terminal GalNAc and fucose. Schwann cells stained for GC with GlcNAc or mannose whereas oligodendroglia stained for GC with the terminal disaccharide Gal-(1 3)-GalNAc and N-glycosidic side chains, especially in presumed Golgi zones, but also in processes continued as the outer myelin sheath. The human myelin sheath in the central segment differed from that of the rat in not staining with lectins specific for fucose and terminal GalNAc. Sialic acid and terminal -Gal were seen in the human central segment but these sugars appeared to bind to astroglial structures rather than to the myelin sheath as in the rat. Astrocytes in both rat and man were stained by two fucose-binding lectins. Several lectins revealed affinity for GC in the neurilemmal sheath, and staining of this structure was stronger in the human specimens. Neurons in the human trigeminal ganglion ranged from unstained to strongly positive for fucoconjugate in cytoplasmic bodies and plasmalemma. Positive ganglion cells gave rise to unmyelinated fibers which also stained for fucoconjugate. Remak fibers and their extensions into the substantia gelatinosa of the human spinal cord stained strongly for content of fucose.The stronger lectin affinity for N-glycosidic core sugars in the peripheral as compared with the central segment suggests that lectins localize P_o protein in peripheral myelin. The reactivity for several sugars in the central segment can possibly be attributed to myelin-associated glycoprotein (MAG) of central myelin, but lectin staining for GalNAc shows in addition a biochemically unrecognized GC with O-glycosidic linked oligosaccharides in myelin. The lectin cytochemistry indicates that the 170 K Dalton glycoprotein with PNA affinity obtained from rat sciatic nerves occurs in nodes of Ranvier.This research was supported by NIH Grants AM-10956, HL-29775 and United Health and Medical Research Foundation of South Carolina, Inc. Grant No. 79     

Clinical studies on cell-mediated immunity in patients with renal cell carcinoma: interleukin-2 and interferon-γ production of lymphocytes

Summary The authors examined interleukin-2 (IL-2) production and interferon (IFN) production of peripheral blood mononuclear cells in 28 patients with renal cell carcinoma and 17 control subjects. The peripheral blood was obtained prior to the initiation of therapeutic procedures. The patients were divided into two groups according to tumor size, 5 cm and >5 cm. The production of IL-2 and IFN was measured by immunoradiometric assay. As a result, in the patients with tumors >5 cm, IL-2 and IFN production was impaired. However, in the patients with tumors 5 cm, IFN production was enhanced, though IL-2 production was not significantly different from that of the control subjects. There was no significant correlation between IL-2 production and IFN production.     

Protein kinase C (α, β, γ) in Pacinian corpuscle

Immunocytochemical demonstration of protein kinase C (PKC) subspecies (, , ) was carried out in Pacinian corpuscles of rat hind feet using monoclonal or polyclonal antibodies against each of these subspecies. The inner core cells and lamellae and the Schwann cell cytoplasm of the nerve fiber innervating the corpuscle were strongly positive for PKC -immunoreactivity (IR). In contrast, the axon terminal and the outer core did not display any positive -IR. Very weak PKC -IR was detected in the ultraterminal region of the axon terminal, while the trunk region showed no immunoreactivity. Very faint PKC -IR was found also in the lamellar cells located at the periphery of the inner core and the endoneurial fibroblasts in the intermediate layer. PKC -IR was not detected in any part of the corpuscle. The strong PKC -IR in the inner core and the presence of absence of PKC -, -, and -IR in the axon terminal are discussed from the point of view of the functional aspects of each part.     

Relations between Computational and Experimental Engineering Approaches to Molecules from Molecular Fragments

Computational techniques have become important tools in the preliminary stages of the design of new molecules. The mutual arrangements of interacting molecular parts and the required accessibility of important reactive groups on the peripheral regions of possible new molecules can be tested by computational means before the expensive and often complex synthetic methods are used to the actual construction of these molecules. There are some common features involved in the computational representation of molecular fragments and the synthetic methodologies used in the process of incorporating actual molecular fragments in such engineered molecules. One trend that appears to link these two approaches is based on the following observation: the greater local autonomy is shown by the calculated electron density contribution of a given molecular fragment, the more likely that this fragment can be regarded as a suitable building block of the molecule, and it is also more likely that there are convenient synthetic methods for the delivery of this fragment to its desired target location in the new molecule to be synthesized. For a precise formulation of this statement, a new concept, the degree of molecular fragment autonomy is introduced, using the inherent properties of molecular electron densities.     

Interaction of cycloalkanoprogesterones with mammalian progesterone receptor: binding of pregna-D'-pentaranes in the calf uterine cytosol

Pregna-D'-pentaranes (pentaranes) are modified progesterones with demonstrable progestational activity and contraceptive effect. We have examined the steroid binding characteristics of the two newly synthesized progesterone analogs, Pentarane A (16, 17-cyclohexanoprogesterone) and Pentarane B (6-methyl, 16, 17-cyclohexanoprogesterone), and studied the nature of their interaction with progesterone receptor (PR) from the chicken oviduct and the calf uterine cytosols. Pregna-D'-pentaranes exhibited no affinity for the chick PR but interacted with the calf uterine PR as did R5020. The pentaranes, however, bound PR less tightly. R5020- or pentarane-bound PR sedimented as an 8S moiety in 8–30% linear glycerol gradients. Thermal transformation of receptor resulted in the reduction of the 8S form, and caused an increase in the binding of R5020-and progesterone-bound PR complexes to DNA-cellulose. The pentarane-bound PR bound poorly, if at all, to DNA-cellulose. Our data suggest that pentaranes exhibit both similarities and differences with natural and synthetic progestins with respect to their interaction with calf uterine PR. The lack of pentarane binding to chicken PR is reminiscent of the general phenomenon that antiprogestins (RU486, ZK98299, and Org 31710 and Org 31806) do not interact with chicken PR. Pentaranes, therefore, represent unique steroid analogs to investigate the molecular mechanism of steroid hormone action.Abbreviations DMSO Dimethyl sulfoxide - DTT Dithiothreitol - E Estradiol - EDTA Ethylene-diaminetetraacetate - F Cortisol - IA Iodoacetamide - MER -mercaptoethanol - MTG Monothioglycerol - NEM N-ethylmaleimide - Org 31710 (6, 11, 17)-11-(4-dimethylaminophenyl)-6 methyl-4, 5-dihydro[estra-4, 9-diene-17, 2(3H')-furna]-3-one - Org 31806 (7, 11, 17)-11-(4-dimethyl-aminophenyl)-7 methyl-4, 5-dihydro[estra-4, 9-diene-17, 2(3H)-furan]-3-one - P Progesterone - Pentarane A 16, 17-cyclohexanoprogesterone - Pentarane B 6-methyl, 16, 17-cyclohexanoprogesterone - PMSF Phenylmethylsulfonyl Fluoride - PR Progesterone Receptor - R5020 17, 21-dimethylpregna-4, 9(10)-diene-3     

Enzymes in cardenolide-accumulating shoot cultures of Digitalis purpurea L.

In contrast to undifferentiated cell suspension cultures of Digitalis lanata, photomixotrophic shoot cultures of Digitalis purpurea accumulate cardiac glycosides in substantial concentrations. They are used to investigate enzymes of the cardenolide pathway. All cardenolides are 5-configurated. The progesterone 5-reductase and the 3-hydroxysteroid-5-oxidoreductase are present in shoot cultures but not in undifferentiated cell cultures. These enzymes provide precursors for cardenolides, whereas the presence of the progesterone 5-reductase, also present in shoot cultures, is discussed with regard to its role in phytosterol biosynthesis and may be attributed to the general steroid pathway. The progesterone 5-reductase had an activity maximum during the early growth period seven days after onset of cultivation, whereas the corresponding progesterone 5-reductase activity was highest on day 11. The maximum cardenolide accumulation was after 24 days. The enzyme activities present in crude extracts from shoot cultures were characterized with regard to their requirements for NADPH and NADH, pH-optimum, temperature optimum, affinity to their substrates and their localization in the cell. The progesterone 5-reductase was purified 769-fold.Abbreviations DW dry weight - FW fresh weight - PVP polyvinylpyrrolidone     

Comparative Modulation by 3α,5α and 3β,5β Neurosteroids of GABA Binding Sites During Avian Central Nervous System Development

Neurosteroids are endogenous Central Nervous System (CNS) compounds which act mainly by allosteric modulation of the GABA_A receptor complex. The presence of a 3-hydroxyl group and a 5-hydrogen atom have been found to be essential structural requirements for biological activity in mammals. In the present work we report the enhancing activity on [³H]GABA binding to its receptor sites in chick optic lobe produced by progesterone metabolites 3-hydroxy,5-pregnan-20-one (3,5-P) and 3-hydroxy,5-pregnan-20-one (3,5-P). Both steroids were found able to enhance [³H]GABA binding along ontogeny, displaying a similar profile at early developmental stages, while in adulthood 3,5-P had greater potency (EC₅₀ 0.22 M) and enhancing effect (E_max: 122%). In adult synaptic membranes, the two compounds displayed a complex interaction with the GABA_A receptor, disclosed by a Schild plot with slope below one and an incomplete displacement of 3,5-P by its 3,5 isomer. Such complexity could be related to the steroidogenic profile in avian CNS, with 5-reduced progesterone metabolites present since early development, while 3,5-P is found only in adulthood. Bearing in mind differences between avian and mammalian steroidogenic profiles and the relevance of 5-steroids in early avian development, we propose that 3,5-P, instead of the classical potent 3,5-steroids, may be the endogenous modulator of GABAergic activity in developing avian brain.     

Interaction sites on phosphorylase kinase for calmodulin

Holophosphorylase kinase was digested with Glu-C specific protease; from the peptide mixture calmodulin binding peptides were isolated by affinity chromatography and identified by N-terminal sequence analysis. Two peptides originating from the subunit, having a high tendency to form a positively charged amphiphilic helix and containing tryptophane, were synthesized. Additionally, a homologous region of the subunit and a peptide from the subunit present in a region deleted in the isoform were also selected for synthesis. Binding stoichiometry and affinity were determined by following the enhancement in tryptophane fluorescence occurring upon 1:1 complex formation between these peptides and calmodulin. Finally, Ca²⁺ binding to calmodulin in presence of peptides was measured. By this way, the peptides 542–566, 547–571, 660–677 and 597–614 have been found to bind specifically to calmodulin.Together with previously predicted and synthesized calmodulin binding peptides four calmodulin binding regions have been characterized on each the and subunits. It can be concluded that endogenous calmodulin can bind to two calmodulin binding regions in as well as to two regions in and . Exogenous calmodulin can bind to two regions in and in . A binding stoichiometry of 0.8mol of calmodulin/ protomer of phosphorylase kinase has been determined by inhibiting the ubiquitination of calmodulin with phosphorylase kinase. Phosphorylase kinase is half maximally activated by 23nM calmodulin which is in the affinity range of calmodulin binding peptides from to calmodulin. Therefore, binding of exogenous calmodulin to activates the enzyme. A model for switching endogenous calmodulin between , and and modulation of ATP binding to as well as Mg²⁺/ADP binding to by calmodulin is presented.     

Localization of Transforming Growth Factor β in Association with Neuromuscular Junctions in Adult Human Muscle

Transforming growth factors- 1, 2, and 3 are known for their regulatory function in embryogenesis, fibrogenesis, and tissue repair of different cell types. A trophic function of TGF- subclasses for motoneurons has been shown in vitro. TGF- 1 is a potent survival factor for cultured embryonic rat motoneurons. In addition, TGF- 1 stimulates proliferation of rat Schwann cells. Recently, TGF- 2 has been reported to be associated with the subsynaptic nuclei of mature rat neuromuscular junctions. In this study, we investigated the expression of TGF- 1, 2, and 3 at neuromuscular junctions in skeletal muscle of 11 adults without neuromuscular disease. On muscle biopsies, neuromuscular junctions were depicted by acetylcholine esterase reaction and acetylcholine receptor antibodies. TGF- 1, 2, and 3 were stained immunohistochemically with monoclonal antibodies. Some muscle fibers showed low levels of inhomogeneous immunoreactivity for both TGF- 1 and TGF- 3. Intense immunoreactivity of TGF- 1 and 3 was shown at the postsynaptic area of neuromuscular junctions. TGF- 2 was expressed in the same subcellular distribution, but less strongly. In conclusion, the colocalization of TGF- with neuromuscular junctions may suggest a significant function in neuromuscular communication.     

Oxidation of 17α,20β- and 17α,20α-Dihydroxypregn-4-en-3-ones,Side Products of Progesterone Biotransformation with Recombinant Microorganisms Expressing Cytochrome P-45017α

Progesterone biotransformation with recombinant yeasts Yarrowia lipolytica E129A15 and Saccharomyces cerevisiae GRF18/YEp5117 expressing bovine adrenocortical cytochrome P-45017 yielded 17-hydroxyprogesterone and two diols, 17,20- and 17,20-dihydroxypregn-4-en-3-ones. The oxidation of mixtures of the three steroids with chromic acid resulted in the cleavage of 17–20 bonds in the diols with the formation of androst-4-ene-3,17-dione. The biotransformation of pregn-4-ene-20-ol-3-one by means of Y. lipolytica E129A15 was accompanied by the following reactions: the primary oxidation of these compounds to progesterone and the subsequent successive reactions of 17-hydroxylation and 20- and 20-reduction. The results widen the possibilities of enzymatic and chemical modifications of steroids.     

Synthesis and secretion of caseins by the mouse mammary gland: Production and characterization of new polyclonal antibodies

Polyclonal antibodies to mouse - and /-caseins were raised in rabbits. These antibodies display tissue- and species specificity as shown by immunoblotting. Immunohistochemical analyses demonstrate that both - and /-caseins were synthesized and secreted from virtually all lactating mammary epithelial cells, in a pattern very similar to that of the mouse -lactalbumin. Residual amounts of caseins were located also in the apical surface of epithelial cells surrounding the ducal lumen of virgin mammary gland sections. In contrast to the significant level of -casein in the milk, the amount of this protein compared to - or -caseins was extremely low in medium conditioned for 24 h by mammary explants of mid-pregnant mice immediately after explantation or after 4 days.     

Large scale preparation of PA-oligosaccharides from glycoproteins using an improved extraction method

We have developed a new method for the large scale preparation of pyridylaminated (PA-) oligosaccharides from glycoproteins. Phenol/chloroform extration was adapted for the removal of protein and excess 2-aminopyridine, improving the efficiency of preparation. From a 2.5 g sample of human apo-transferrin, 25–30 mol of agalacto biantennary PA-oligosaccharide could be obtained. By increasing the concentration of PA-oligosaccharide substrate, we were able to detect a very low level ofN-acetylglucosaminlytransferase IV activity in CHO cell extracts.Abbreviations PA 2-aminopyridine - SDS sodium dodecyl sulfate - GlcNAc N-acetylglucosamine - GnT N-acetylglucosaminyltransferase - Gn,Gn-bi-PA GlcNAc1-2Man1-3(GlcNAc1-2Man1-6)Man1-4GlcNAc1-4GlcNAc-2-aminopyridine - Gn,Gn,Gn-tri-PA GlcNAc1-2(GlcNAc1-4)Man1-3(GlcNAc1-2Man1-6)Man1-4GlcNAc1-4GlcNAc-2-aminopyridine - Gn,Gn,Gn-trí-PA GlcNAc1-2Man1-3({GlcNAc1-2(GlcNAc1-6)Man1-6})Man1-4GlcNac1-4GlcNAc-2-aminopyridine - Gn,(Gn),Gn-bi-PA GlcNAc1-2Man1-3(GlcNAc1-4)(GlcNAc1-2Man1-6)Man1-4GlcNAc1-4GlcNAc-2-aminopyridine     

Adoptions expérimentales de larves entre des fourmis de genres différents:Leptothorax nylanderi Förster etSolenopsis fugax Latreille

Résumé En l'absence de son propre couvain,Solenopsis fugax a élevé des larves deLeptothorax nylanderi, à la température de 22°C. Les ouvrières deSolenopsis détruisirent une partie de ces larves mais nourrirent celles qu'elles épargnèrent; ces dernières grossirent lentement pendant cinq à six mois, sans atteindre le stade prénymphe. Lorsque les ouvrières deS. fugax et les larves deL. nylanderi furent soumises ensemble à un hivernage préalable, elles donnèrent les mêmes résultats que sans hivernage. La présence d'une jeune reine deSolenopsis fut défavorable aux larves deLeptothorax.Inversement,L. nylanderi fut capable d'élever, à la température de 22°C, des larves deS. fugax et de les amener jusqu'au stade adulte. En présence de leurs propres larves, les ouvrières deL. nylanderi détruisirent tapidement toutes les larves deS. fugax introduites dans leur nid. D'autre part, un jeune couvain deLeptothorax remplaçait plus ou moins rapidement les larves deLeptothorax enlevées au préalable; sa présence était alors défavorable au développement des larves deSolenopsis. Un hivernage en début d'expérience fut plutôt favorable auxS. fugax, de même que la présence d'une reine féconde deLeptothorax. LesSolenopsis ainsi obtenus n'ont pas vécu plus de sept semaines. Ils étaient tous de caste ouvrière et de taille très petite.

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Summary When its own eggs and larvae missed,Solenopsis fugax bred larvae ofLeptothorax nylanderi, at a temperature of 22°C. TheSolenopsis workers killed some of this larvae and fed the others; these slowly grew bigger during five or six months but never reached the pre-pupa stage. The result was the same if the workers ofS. fugax and the larvae ofL. nylanderi overwintered together or not at all. A youngSolenopsis queen being there was noxious to the larvae ofLeptothorax.On the contrary,L. nylanderi has been able to breed larvae ofS. fugax up to the imago stage, at a temperature of 22°C. When its own larvae were in the nest, together with larvae ofS. fugax, the workers ofL. nylanderi killed the larvae ofS. fugax. On the other hand, new eggs and young larvae ofLeptothorax had to replace, more or less quickly, the larvae which had been taken away, and that was noxious to the growth ofSolenopsis larvae. An overwintering at the beginning of the experiment was rather favourable toS. fugax as was the presence of a fecundLeptothorax queen. TheSolenopsis thus obtained lived no longer than seven weeks. They all were workers and very small.

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S. Fugax L. Nylanderi 22° . Leptothorax , , , , . . S. Fugax Leptothorax.,L. Nylanderi 22° S. Fugax . L. Nylanderi ( )Leptothorax ; S. Fugax Solenopsis, Leptothorax. S. Fugax . .