Gamma-crystallins of the human eye lens: expression analysis of five members of the gene family.

While only two gamma-crystallins have been identified in the human eye lens, molecular studies indicate that the human gamma-crystallins are encoded in a multigene family comprising at least seven closely related members. Sequence analysis of five of these genes has suggested that three (gamma 1-2, G3, and G4) are potentially active, while two (G1 psi and G2 psi) correspond to closely related pseudogenes. Here we report on the detailed structure of a sixth gamma-crystallin gene, G5, and our results obtained with transient expression assays to characterize both the promoter activity and translation products of five members of the gene family. We show that 5'-flanking sequences of G1 psi and G2 psi lacked detectable promoter activity, while the corresponding sequences of G3, G4, and G5 were able to direct high levels of expression of the bacterial chloramphenicol acetyltransferase gene in primary lens epithelia, but not in cultures of nonlens origin. Detailed sequence comparisons indicated that active genes contained several conserved sequence tracts 5' of the TATA box which may constitute functional elements of a lens-specific gamma-crystallin promoter. Expression of the gamma-crystallin coding sequences from the human metallothionein IIA promoter in nonlens cells facilitated characterization of the polypeptides encoded by individual gamma-genes and, in future studies, should permit comparison of these proteins with distinct gamma-crystallins in the human lens.     

Responses of pyriform cortex neurons to excitatory amino acids: Voltage dependence,conductance changes,and effects of divalent cations

The actions of ionophoretically applied N-methyl aspartate (NMA), quisqualate, and kainate, thought to activate three different types of excitatory amino acid receptors, were studied on pyramidal neurons of the rat pyriform cortex, maintained in an isolated, submerged, and perfused brain slice. Intracellular recordings were made with either K acetate or CsCl electrodes. In most neurons all three agonists elicited monophasic responses which could be evoked at 20-sec intervals. Some neurons showed biphasic responses, most commonly to kainate but, on occasion, also for quisqualate. The slower component appeared to be correlated with excitotoxicity and, consequently, was difficult to study. As a result the kainate responses studied were from neurons selected for having a single component. In neurons selected for having a linear current-voltage relationship or neurons loaded with Cs to suppress K conductance and linearize the current-voltage relationship, the average changes in resistance recorded during ionophoretic responses at resting potential were as follows: NMA, 131.2 +/- 6.7% of control; kainate, 104.7 +/- 5.8% of control; and quisqualate, 92.8 +/- 2.8% of control. The magnitude and direction of the conductance change were very reproducible in any one neuron, but especially for kainate some cells showed clear conductance increases, while others showed clear conductance decreases. Using CsCl electrodes it was possible to reduce K+ conductance and depolarize the neurons over a wider range. By passing depolarizing current it was possible to reverse the responses. The response to all three agonists reversed at the same depolarized potential. This observation indicates that while there are differences in the ionic channels associated with the three agonists at resting potential, the channels have similar properties at more depolarized potentials. Responses to all three agonists were influenced by the concentrations of divalent cations in the perfusion medium. The NMA responses were most sensitive to Mg, increasing in amplitude in the absence of Mg and being depressed by Mg elevation. All responses were sensitive to Ca, with discharges being greatly increased by low Ca and depressed by high Ca. The kainate response was most sensitive to Ca concentration changes. Unlike reports from other preparations the apparent conductance decreases to NMA were not altered by the perfusion of solutions with either no added Mg or no added Ca. The NMA response was very much reduced in either Co (1-2 mM) or Zn (100-200 microM).(ABSTRACT TRUNCATED AT 400 WORDS)     

Fucosyltransferase expression in human platelets and leucocytes

The activities of -2-l-fucosyltransferase and -3-l-fucosyltransferase were measured in human platelets and leucocytes from normal donors, -2-l-Fucosyltransferase was found in platelets but not in leucocytes. In contrast -3-l-fucosyltransferase was not detected in platelets but was present in leucocytes where it was demonstrated in the neutrophil, monocyte and lymphocyte fractions.     

Electrically evoked contractions of the triceps surae during and following 21 days of voluntary leg immobilization

The effects of 21 days voluntary leg (plaster) immobilization on the mechanical properties of the triceps surae have been studied in 11 young female subjects, mean age 19.4 years. The results show that during the period of immobilization the mean time to peak tension (TPT) and half relaxation time (1/2RT) and tension (Pt) of the maximal twitch increased significantly (p less than 0.001) but the effects were short lived. Maximal tension and contraction times of the twitch recovered within 2-14 days following the removal of the plaster cast. The electrically evoked tetanic tensions at 10 Hz and 20 Hz did not change significantly (P greater than 0.1) during immobilization, but the 50 Hz tetanic tension (Po50) and maximal voluntary contraction (MVC) were reduced (p less than 0.05). The fall in Po50 and MVC was associated with 10% decrease in the estimated muscle (plus bone) cross-sectional area. The relative (%) change in Po50 and MVC following immobilization was related to the initial physiological status (as indicated by the response of the triceps surae to a standard fatigue test prior to immobilization) of the muscle. The rate of rise and recovery fall of the tetanus were slightly but significantly (p less than 0.01) reduced on day 7 of immobilization, but thereafter remained constant. The isokinetic properties of the triceps surae as reflected in the measured torque/velocity relation of the muscle in 4 subjects did not change significantly if account was taken of the slight degree of atrophy present following immobilization.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mapping of the gene for anti-müllerian hormone to the short arm of human chromosome 19

The gene coding for human anti-Müllerian hormone (AMH) was localized to subbands p13.2----p13.3 on chromosome 19, using in situ hybridization and Southern blot analysis of a panel of man-mouse and man-hamster somatic cell hybrids.     

Neuroanatomy of morphine-modulating peptides

Antisera against two mammalian peptides related to the molluscan cardioexcitatory peptide Phe-Met-Arg-Phe-NH2 were used to locate immunoreactive neurons in the rat brain, nerve fibres and terminals in the spinal cord, sympathetic ganglion cells and adrenal chromaffin cells. Immunoreactivity for the newly characterised octa- and octadecapeptide was detected in nerve cell bodies in the hypothalamic area, including parts of the dorsomedial, periventricular and paraventricular nuclei, and in the nucleus tractus solitarii. Nerve terminals in the superficial laminae of the spinal cord were also immunoreactive for these peptides, while the sensory ganglia were nonreactive. Some principal ganglion cells in the superior cervical ganglia exhibited bright immunofluorescence for the peptides, and a few adrenal medullary cells were immunoreactive. The presence of these peptides in the substantia gelatinosa of the spinal cord suggests that they may be involved in sensory neurotransmission, especially in the mechanisms mediating pain. In the hypothalamo-hypophysial system these peptides may be involved in the regulation of hormonal systems. They may also act as co-transmitters in the sympathetic nervous system.     

Ultrastructural immunocytochemical study of the massa caudalis of the subcommissural organ-Reissner's fiber complex in lamprey larvae (Geotria australis): Evidence for a terminal vascular route of secretory material

Summary The massa caudalis of the subcommissural organ-Reissner's fiber complex of lamprey larvae (Geotria australis) was studied immunocytochemically at the ultrastructural level by use of the immunoperoxidase-silver methenamine procedure. An antiserum raised against bovine Reissner's fiber was utilized as primary antibody.The caudalmost portion of the central canal and its ampulla caudalis communicate, via wide intercellular spaces in their dorsal wall, with large cavities or lacunae. In addition, distinct openings in the dorsal wall of the ampulla establish an open communication between the latter and the lacunae. The lacunae are lined by slender processes of cells of unknown nature. No junctional complexes can be observed between these cells, which lack a basal lamina. The lacunae communicate with structures resembling blood capillaries, however, they are devoid of a basal lamina. These peculiar vessels, in turn, are in direct communication with characteristic blood capillaries.Reissner's fiber (RF) and its massa caudalis are strongly immunoreactive with the antiserum used. The wide intercellular spaces in the dorsal wall of the central canal and the ampulla, as well as the lumina of the (i) lacunae, (ii) modified vessels and (iii) blood capillaries are filled with a flocculent, strongly immunoreactive material. No immunoreactive material was found outside these structures. Thus, the blood capillaries appear to represent the only final target of RF-material arriving at the ampulla caudalis.Supported by Grant I 38259 from the Stiftung Volkswagenwerk, Federal Republic of Germany, Grant S-85-39 from the Dirección de Investigaciones, Universidad Austral de Chile, and Grant 6027 from Fondo Nacional de Desarrollo Científico y Tecnológico, Chile. The authors express their gratitude to Mrs. Elizabeth Santibáñez and Mr. Julio Lamilla for providing the lamprey larvae and to Mr. Humberto Molina for preparing the three-dimensional drawing     

A morphometric and ultrastructural study of lithium-induced changes in the medullary collecting ducts of the rat kidney

Summary Rats were given a lithium-containing diet (40 mmol/kg) to Study the effect of lithium on the structure of collecting ducts from the inner stripe of the outer medulla. The results show that there is a significant increase in the volume density of collecting ducts already after one week on this diet. The volume density of both intercalated and principal cells increases, whereas the volume density of mitochondria in the cytoplasm increases in the intercalated cells only. The increased volume of both principal and intercalated cells seems to be part of a general hyperplasia and hyperactivity of the collecting duct, which may in some way be related to the effects of lithium on vasopressinmediated water transport. The specific changes in the intercalated cells may be a consequence of the effects of lithium on distal nephron potassium and hydrogen ion transport in the distal nephron.     

Cell wall mannoproteins during the population growth phases in Saccharomyces cerevisiae

Mannoproteins from cell walls of Saccharomyces cerevisiae synthesized at successive stages of the population growth cycle have been solubilized with Zymolyase and subsequently analyzed. The major change along the population cycle concerned a large size mannoprotein material; the size of the newly-synthesized molecules varied from 120,000–500,000 (mean of about 200,000) at early exponential phase to 250,000–350,000 (mean of about 300,000) at late exponential phase. These differences are due to modifications in the amount of N-glycosidically linked mannose residues, since the size of the peptide moiety was 90,000–100,000 at all growth stages and the level of O-glycosylation changed only slightly. After, incubation of the purified walls with concanavalin A-ferritin and subsequent analysis by electron microscopy, labelling was localized at the external and internal faces of the walls. The middle space of these was labelled after digestion of the glucan network with Zymolyase, which demonstrate the presence of mannoproteins in close contact with the structural glucan molecules throughout the wall.Abbreviations BSA bovine serum albumin - Con A concanavalin A - SDS sodium dodecyl sulphate     

Chemical structures of a galactose-rich glycoprotein of Leishmania tarentolae

1. Aqueous phenol treatment of water extracted disrupted cells of Leishmania tarentolae (LV-414) provided a glycoprotein mixture which was purified by gel filtration chromatography, and Concanavalin A-Sepharose column. 2. The bound fraction on Concanavalin A-Sepharose column (protein 74%, and carbohydrate, 26%) had [alpha]D + 9 degrees and contained mannose (18%), galactose (60%), and glucose (22%), and some of the galactose residues were resistant to periodate oxidation. 3. Treatment of the phenol extract with hot aqueous NaBH4 containing NaOH gave a preparation having mannose (12%), galactose (82%), and glucose (6%). 4. Methylation analysis showed the presence of a mainly linear structure with non-reducing end-units of mannopyranose (6%), 3-O-substituted galactopyranosyl (64%), 2-O- (11%), and 6-O- (5%) substituted mannopyranosyl, and 4-O- (9%), and 4,6-di-O- (3%) substituted glycopyranosyl units. 5. The specific rotation of the preparation, +20 degrees, indicated beta-linked galactopyranosyl units.