Ultrastructure of the parathyroid gland of fetal and pregnant golden hamsters subjected to hypergravity environment.

The ultrastructure of the parathyroid glands of fetal and pregnant golden hamsters exposed to 5-gravity environment was studied. In the centrifuged fetal animals the Golgi complexes associated with some prosecretory granules were significantly increased compared with those of the control fetal animals, and several secretory granules were located in a peripheral position just beneath the plasma membrane. In the centrifuged pregnant animals the Golgi complexes associated with numerous prosecretory granules were significantly increased, cisternae of the granular endoplasmic reticulum appeared to be increased compared with those of the control pregnant animals, many secretory granules were located in the peripheral cytoplasm and several granules were situated close to the plasma membrane. These findings suggest that in the parathyroid glands of the fetal golden hamsters as well as the pregnant animals the synthesis and release of parathyroid hormone may be stimulated in response to a hypergravity environment.     

Effects of hypergravity environment on the parathyroid gland of the norepinephrine-treated golden hamster: a stereological study.

Effects of 5-gravity environment on the ultrastructure of the parathyroid glands of norepinephrine-treated golden hamsters were studied. In the centrifuged animals treated with norepinephrine, the volume density occupied by the Golgi complexes associated with numerous prosecretory granules was significantly increased compared with that of the control, centrifuged and norepinephrine-treated animals, as well as the volume density occupied by the cisternae of the granular endoplasmic reticulum compared with that of the control and centrifuged animals. In addition, in the centrifuged animals treated with norepinephrine, numerous secretory granules were situated close to the plasma membrane. It is suggested that the synthesis and release of secretory granules may be markedly stimulated in the parathyroid glands of the norepinephrine-treated golden hamsters subjected to a hypergravity environment.     

Age-associated oxygen damage and mutations in mitochondrial DNA in human hearts.

Some mutations in mitochondrial DNA (mtDNA) causing a number of neuromuscular diseases are suggested to arise spontaneously during the life of an individual. To substantiate the extent and the rate of these somatic mutations, mtDNA specimens from post-mortem human heart muscles of subjects in differing age groups were hydrolyzed. 8-Hydroxy-deoxyguanosine (8-OH-dG), a hydroxyl-radical adduct of deoxyguanosine, in mtDNA, was quantitatively determined using a micro high-performance liquid chromatography/mass spectrometry system. In each specimen, the mtDNA with a 7.4 kilo base-pair deletion was quantified by the kinetic polymerase chain reaction method. In association with age, the 8-OH-dG content accumulated exponentially up to 1.5% with a correlative increase in the content of the deleted mtDNA up to 7%. Clear correlation between the 8-OH-dG content in mtDNA and the population of mtDNA with a deletion (r = 0.93, P < 0.01) gives insight into the mechanism for the generation of a large deletion. These results indicate that accumulation of somatically acquired oxygen damage together with age-associated mutations in mtDNA which lead to bioenergetic deficiency and the heart muscle weakness are inevitable in human life.     

Expression of human T-cell leukemia virus type I protease in Escherichia coli.

Human T-cell leukemia virus type I (HTLV-I) genome is believed to encode its own protease, although the protease has not yet been detected. To identify the HTLV-I protease, an in-frame gag (3' portion)-prt region was expressed in Escherichia coli. The 14-kDa product was detected using antisera against a synthetic peptide mimicking the fragment of HTLV-I protease, although the molecular weight of the primary translational product was 27,000. A cell extract had a proteolytic activity to cleave a synthetic peptide substrate containing the cleavage site of gag p19/p24 at the correct site in vitro. Replacement of the putative active site Asp-64 with Gly abolished both in vivo processing activity and in vitro proteolytic activity. These results suggest that the 14-kDa product is the mature enzymatically active HTLV-I protease generated through posttranslational autoprocessing in E. coli.     

Purification and some properties of the small subunit of cytochrome b558 from human neutrophils

We have attempted to purify the heme moiety of cytochrome b558 from human neutrophils. Cytochrome b558 was solubilized from the crude membrane fraction which was pretreated with both 1 M potassium phosphate buffer and 1% octyl glucoside at low ionic strength. The solubilization of cytochrome b558 was carried out efficiently with 1.6% octyl glucoside in the presence of 100 mM phosphate buffer. Solubilized cytochrome b558 was purified by hydroxylapatite, DEAE-Sephacel, and Mono Q fast protein liquid chromatography. The specific content of purified cytochrome b558 was 37 nmol/mg of protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of purified cytochrome b558 revealed a single band of 20,000 Da. The large subunit of cytochrome b558, which has been reported by others, could not be found in purified cytochrome b558 even with silver staining. The amino acid composition of the heme-containing moiety of cytochrome b558 was abundant in hydrophobic amino acids. The circular dichroism spectra of both oxidized and reduced b558-type cytochromes exhibited bilobed bands with wavelengths of crossover points closely corresponding to those of the maxima in the optical absorbance spectra at the Soret region. Furthermore, there were some differences in the shoulders and peak widths of CD spectra between oxidized and reduced b558-type cytochromes. These results indicate that this method provides the purification of the small subunit of human cytochrome b558 which is the heme-carrying subunit of cytochrome b558, and suggest that cytochrome b558 has heme-heme interaction and some conformational changes in the alternation of the redox state.     

Isolation and characterization of a new 36-kDa microfibril-associated glycoprotein from porcine aorta

A new connective tissue protein of 36 kDa has been purified from porcine aorta. The biochemical and immunological properties of the protein are distinct from those of microfibril-associated proteins reported previously such as lysyl oxidase, 31-kDa microfibril-associated glycoprotein, and fibrillin. It could bind to concanavalin A-Sepharose and gelatin-Sepharose. The protein contained the sequence Arg-Gly-Asp-Ala in the amino-terminal region, which is the site for the association with cell and extracellular matrix. Using specific antibody raised to the protein, we demonstrated its restricted localization in aorta adventitia. Immunoelectron microscopy specified its location to a class of extracellular structural elements described as elastin-microfibrils.     

Functional and phenotypic alterations in T cell subsets during the course of MAIDS, a murine retrovirus-induced immunodeficiency syndrome

The functional and phenotypic characteristics of Ly-4(CD4)+ and Ly-2(CD8)+ T cells were studied after induction of murine AIDS with LP-BM5 murine leukemia virus. Assays of spleen cells for their ability to generate in vitro CTL responses to TNP-modified autologous cells (self + x CTL) and to alloantigens (allo CTL) showed that self + x CTL responses were greatly impaired at 3 to 4 wk postinfection and were undetectable thereafter. Allo CTL responses were normal at 3 to 4 wk, but were reduced at 8 to 9 wk and absent at 14 wk postinfection. This sequential loss of self + x and allo CTL responses was related to a selective defect in Ly-4(CD4)+ Th cell function associated with impaired production of IL-2 and deficient proliferative responses to Con A or to soluble Ag. Changes in the functional characteristics of Ly-4(CD4)+ T cells were unrelated to changes in their frequency in spleen, but did correlate with marked alterations in their distribution among four subsets defined by mAb SM3C11 and SM6C10. Assays of CTL responses generated by mixtures of spleen cells from normal and infected mice suggested that active suppression of Ly-4(CD4)+ Th function may contribute to this defect. Studies of Ly-2(CD8)+ T cells showed that infection with LP-BM5 murine leukemia virus also induced a major phenotypic shift in subpopulations defined by their reactivity with mAb 6C10. However, this phenotypic change did not appear to correlate with major functional defects.     

A single VH gene is utilized predominantly in anti-BrMRBC hybridomas derived from purified Ly-1 B cells. Definition of the VH11 family

By establishing hybridomas from two distinct surface IgM+ splenic B cell populations, Ly-1 B cells and "conventional" (Ly-1-) B cells, we found that the Ly-1 B population includes a 30 to 70 times higher frequency (1 to 2%) of cells with specificity for bromelain treated autologous red blood cells (anti-BrMRBC) when compared with conventional B cells (0.03%). We cloned and sequenced the V genes encoding anti-BrMRBC antibody from two hybridomas made with Ly-1 B cells sorted from the spleen of SM/J mice. The VH sequence (for both) is identical with the previously reported sequence associated with this specificity and belongs to a new VH gene family. This gene family, defined here as VH11, has only two members and is the predominant VH rearranged in a collection of Ly-1 B derived anti-BrMRBC hybridomas, always in association with a single VL gene (a member of the V kappa 9 family). Furthermore, analysis of hybridomas made with Ly-1 B cells sorted from the peritoneum reveals a yet higher increased frequency of VH11-encoded anti-BrMRBC specificity (30%). This variation in frequency of anti-BrMRBC in the Ly-1 population depending on location, together with the repeated association of VH11 with a particular V kappa gene suggest that antigen driven selection is (at least in part) responsible for the biased V gene expression seen in this population. Furthermore, a mechanism that might contribute to biased expression, preferential rearrangement due to close proximity to J (as seen in pre-B lines), is excluded by localization of VH11 5' to several of the more J-proximal families (Q52, 7183).