Production and characterization of monoclonal antibodies to the mammalian sperm cytoskeleton

The cytoskeleton exerts a direct effect on the function of sperm by influencing the distribution of subcellular organelles and plasma membrane molecules. We have prepared six monoclonal antibodies to Triton X-100-insoluble components of the bull sperm cytoskeleton. One of the antibodies reacts with a detachable portion of the bull sperm acrosome. The remainder include an antibody that recognizes the principal and end piece of the tail and another that is specific to the middle piece. Two of the antibodies yield dissimilar staining patterns of the neck region and the tail, and the final monoclonal antibody stains the subacrosomal region and a detachable acrosomal domain of bull sperm. The cross reactivities of the antibodies with hamster sperm and PtK2 cells are described, as is the recognition of bull sperm polypeptides on western blots. The results suggest that these antibodies will provide interesting insights concerning the role of the cytoskeleton in sperm development and function.     

Expression of mammalian liver glycolytic/ gluconeogenic enzymes in Escherichia coli: Recovery of active enzyme is strain and temperature dependent

A number of mammalian enzymes have been expressed in Escherichia coli using the T7 RNA polymerase system, but the production of large amounts of these proteins has been limited by the low percentage of active enzyme that is found in the soluble fraction. In this report the effect of induction temperature was tested on the recovery of four rat liver enzymes, 6-phosphofructo-2-kinase/fructose-2,6- bisphosphatase, fructose-2,6-bisphosphatase, glucokinase, and fructose-1,6-bisphosphatase. We also tested the effect using a host cell strain that contains a plasmid encoding T7 lysozyme, an inhibitor of T7 RNA polymerase. Large amounts of the first three enzymes accumulated in the cells after 4 h of induction at 37 degrees C, but only about 1-2% of the total expressed proteins were recovered in a soluble, active form. When the induction was carried out at 22 degrees C for 48 h with the pLysS strain, 20- to 30-fold higher amounts of the active expressed enzymes were recovered in the soluble fraction, even though the total accumulation and the rate of synthesis of these proteins were reduced. The optimal concentration of isopropyl-1-thio-beta-D-galactopyranoside required for induction was the same at both temperatures. On the other hand, the recovery of active fructose-1,6-bisphosphatase, a heat-stable enzyme, was 66% at 37 degrees C and was essentially unchanged at an induction temperature of 22 degrees C. Lowered induction temperature would appear to be of utility for enhanced recovery of active mammalian enzymes which are insoluble in E. coli cytosol at 37 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

The Supramolecular Structure of Photosystem II — Phycobilisome-Complexes of Porphyridium cruentum

The structure and arrangement of phycobilisomes of the unicellular red alga Porphyridium cruentum is compared with the organization of the thylakoid freeze-fracture particles in order to determine the relationship between phycobilisomes and photosystem II. The hemi-ellipsoidal phycobilisomes, 20 nm thick, are predominantly organized into rows; their centre to centre periodicity is 30–40 nm, so that they are well separated by a gap of 10–20 nm. The phycobilisomes are cleaved by a central faint furrow, parallel to the long axis from top to base. The organization of the exoplasmic particles in rows is similar to the arrangement of the phycobilisomes so that a structural relationship between both systems, previously demonstrated in cyanobacteria, is evident. Within the rows, the 10 nm EF-particles are grouped in tetrameric complexes separated by distances similar to those observed for phycobilisomes. We propose that the tetrameric EF-particle complexes correspond to tetrameric photosystem II complexes which bind one hemi-ellipsoidal phycobilisome on the stroma exposed surface of the thylakoid. A hypothetical model of this photosystem II-phycobilisome complex is presented.     

Mechanosensitive Properties of BK Channels from Embryonic Rat Neuroepithelium

The mechanosensitive properties of large-conductance Ca²⁺-activated K⁺ (BK) channels from embryonic rat neuroepithelium were investigated with the cell-attached and inside-out configurations of the patch-clamp technique. The channels were activated in both recording configurations by negative pressures applied to the patch electrode, but reversal of the effect was total and immediate in inside-out patches whereas it was incomplete and delayed in on-cell patches. This mechanosensitivity was not mediated by Ca²⁺ ions or fatty acids, suggesting that it is an intrinsic property of these channels. Cytochalasin B did not affect mechanosensitivity in on-cell patches but increased it in inside-out patches. Kinetic studies showed that stretch increased the mean open time of the channels and decreased the slowest time constant of their closed-time distributions. The present as well as previous results suggest complex interactions between embryonic BK channels and their membranous and submembranous environment. Received: 1 February 1996/Revised: 25 March 1996     

In Vivo and In Vitro Evidence for the Biosynthesis of Testosterone in the Telencephalon of the Female Frog

Abstract: Neurons and glial cells are capable of synthesizing various steroid hormones, but biosynthesis of testosterone in the CNS has never been reported. The aim of the present study was to demonstrate the synthesis of testosterone in the frog brain. The presence of 17β-hydroxysteroid dehydrogenase (17β-HSD)-like immunoreactivity was detected in a population of glial cells located in the telencephalon. Reversed-phase HPLC analysis of brain tissue extracts combined with radioimmunoassay detection revealed the presence of substantial amounts of testosterone and 5α-dihydrotestosterone (5α-DHT) in the telencephalon where 17β-HSD-positive cells were visualized. In male frogs, castration totally suppressed testosterone and 5α-DHT in the blood and in the rhombencephalon but did not affect the concentration of these two steroids in the telencephalon. Chemical characterization of testosterone in female frog telencephalon extracts was performed by coupling HPLC analysis with gas chromatography-mass spectrometry. Using the pulse-chase technique with [³H]pregnenolone as a precursor, the formation of a series of metabolites was observed, including dehydroepiandrosterone, androstenedione, testosterone, 5α-DHT, and estradiol. These data demonstrate the existence of an active form of 17β-HSD in the frog telencephalon, which is likely involved in testosterone biosynthesis within the brain.     

The association of proctolin with a ventral abdominal muscle of Locusta migratoria

The association of proctolin with the external ventral protractor muscle of the VIIth abdominal segment (M234) of Locusta migratoria was investigated using immunohistochemistry and RP-HPLC in conjunction with the sensitive locust oviduct bioassay. Immunohistochemistry of whole-mount tissues revealed two proctolin-like immunoreactive axons in N2B_2b1 (the nerve branch which innervates M234) as well as immunoreactive processes and varicosities on the surface of M234. Immunogold staining of M234 demonstrated that the proctolin-like immunoreactivity was present in electron-dense granules in its motor terminals. A material indistinguishable from proctolin and with proctolin-like bioactivity co-eluted with authentic proctolin on two different RP-HPLC systems. Bath application of proctolin at concentrations greater than 10^-11 M resulted in a dose-dependent increase in the neurally-evoked fast twitch amplitude and duration of M234. Concentrations greater than 10^-9 M resulted in a dose-dependent increase in basal tonus of M234. These results indicate that proctolin, or a peptide very similar to proctolin, is present in the motor innervation of M234 and acts as a cotransmitter and/or neuromodulator at this typical fast skeletal muscle.Abbreviations M234 external ventral protractor muscle of the Vllth abdominal segment - RP-HPLC reverse-phase high pressure liquid chromatography - TFA trifluoroacetic acid     

Identification of a subdomain within DNA-(cytosine-C5)-methyltransferases responsible for the recognition of the 5' part of their DNA target.

In previous work on DNA-(cytosine-C5)-methyltransferases (C5-MTases), domains had been identified which are responsible for the sequence specificity of the different enzymes (target-recognizing domains, TRDs). Here we have analyzed the DNA methylation patterns of two C5-MTases containing reciprocal chimeric TRDs, consisting of the N- and C-terminal parts derived from two different parental TRDs specifying the recognition of 5'-CC(A/T)GG-3' and 5'-GCNGC-3'. Sequences recognized by these engineered MTases were non-symmetrical and degenerate, but contained at their 5' part a consensus sequence which was very similar to the 5' part of the target recognized by the parental TRD which contributed the N-terminal moiety of the chimeric TRD. The results are discussed in connection with the present understanding of the mechanism of DNA target recognition by C5-MTases. They demonstrate the possibility of designing C5-MTases with novel DNA methylation specificities.     

Selection of monosomic addition plants in offspring families using repetitive DNA probes in Beta L.

The distribution of two repetitive DNA probes Sat-121 and PB6-4, specific for the section Procumbentes of the genus Beta, was tested in 16 B. patellaris monosomic addition families using a dot-blot hybridization procedure. All monosomic additions were accurately distinguished from diploid sib plants with both DNA probes. The probe PB6-4, with the strongest signal after hybridization, was selected for rapid screening of an extensive number of putative monosomic additions in B. patellaris or B. procumbens addition families using a squash-blot hybridization procedure. The probe PB6-4 detected 118 monosomic additions in 640 plants (18.4%) in eight different B. procumbens addition families. The addition family with chromosome 4 of B. procumbens was semi-lethal and could not be tested. The distribution of PB6-4 in B. patellaris addition families was confirmed in 63 addition families using the squash-blot procedure. In 4580 plants of these addition families, 628 individual monosomic additions (13.7%) were found. The relationship of the morphological characteristics of monosomic addition plants to the results of the squash-blot hybridization (plants with signal) using probe PB6-4 is quite rigorous but not complete. The correlation between plants with a signal and chromosome number (2n=19) is complete. These results indicate that sequences present on PB6-4 are probably present on all chromosomes of B. patellaris and B. procumbens. The possibility of utilizing the sequence information of Sat-121 for a PCR-based assay to screen for putative monosomic addition plants was also investigated as an alternative to chromosome counting. The DNA-amplification profiles using the primers REP and REP.INV clearly distinguished monosomic addition plants from their diploid sibs.     

A mobile cage facilitates periodic social contact and exercise for singly caged adult Vervet monkeys

Abstract: A mobile exercise cage that expands the quantity and improves the quality of the space available to singly caged adult Vervet monkey males is described. It was easily fitted into an existing caging system and the addition of a resident consort female made it possible for the males to mate and have regular social contact.     

Telomerase activation in mouse mammary tumors: lack of detectable telomere shortening and evidence for regulation of telomerase RNA with cell proliferation.

Activation of telomerase in human cancers is thought to be necessary to overcome the progressive loss of telomeric DNA that accompanies proliferation of normal somatic cells. According to this model, telomerase provides a growth advantage to cells in which extensive terminal sequence loss threatens viability. To test these ideas, we have examined telomere dynamics and telomerase activation during mammary tumorigenesis in mice carrying a mouse mammary tumor virus long terminal repeat-driven Wnt-1 transgene. We also analyzed Wnt-1-induced mammary tumors in mice lacking p53 function. Normal mammary glands, hyperplastic mammary glands, and mammary carcinomas all had the long telomeres (20 to 50 kb) typical of Mus musculus and did not show telomere shortening during tumor development. Nevertheless, telomerase activity and the RNA component of the enzyme were consistently upregulated in Wnt-1-induced mammary tumors compared with normal and hyperplastic tissues. The upregulation of telomerase activity and RNA also occurred during tumorigenesis in p53-deficient mice. The expression of telomerase RNA correlated strongly with histone H4 mRNA in all normal tissues and tumors, indicating that the RNA component of telomerase is regulated with cell proliferation. Telomerase activity in the tumors was elevated to a greater extent than telomerase RNA, implying that the enzymatic activity of telomerase is regulated at additional levels. Our data suggest that the mechanism of telomerase activation in mouse mammary tumors is not linked to global loss of telomere function but involves multiple regulatory events including upregulation of telomerase RNA in proliferating cells.