Analysis of developmental switching in transgenic mice with 5′ and 3′ deletions in the human β globin gene

Our interest in thecis-acting elements that promote the up-regulation of the globin gene has led to a systematic deletion analysis of portions of the globin gene in the context of the HS2 and globin gene using transgenic mice. In constructs that delete the 5 region to only 265 bp, high-level erythroid-specific expression was observed. Further deletion to 122 bp, however, results in significantly reduced expression levels A substitution of a minilocus control region for the single HS2 site was also produced, resulting in increased globin expression over that seen with the HS2 alone. These results are consistent with the presence of an enhancer-like element between –122 and –265. In addition, a construct in which the entire globin gene promoter was replaced by a thymidine kinase promoter was tested. Interestingly, no expression was detected in these transgenic mice. This may indicate the requirement for an erythroid-specific promoter to drive this gene. Finally, the 3 region of the globin gene was deleted in order to examine the effect of a previously defined 3 enhancer region. With deletion of this region, the expression of the human globin gene in transgenic mice is unchanged relative to the parental constructs.     

Functional molecular aspects of the NADH dehydrogenases of plant mitochondria

There are multiple routes of NAD(P)H oxidation associated with the inner membrane of plant mitochondria. These are the phosphorylating NADH dehydrogenase, otherwise known as Complex I, and at least four other nonphosphorylating NAD(P)H dehydrogenases. Complex I has been isolated from beetroot, broad bean, and potato mitochondria. It has at least 32 polypeptides associated with it, contains FMN as its prosthetic group, and the purified enzyme is sensitive to inhibition by rotenone. In terms of subunit complexity it appears similar to the mammalian and fungal enzymes. Some polypeptides display antigenic similarity to subunits fromNeurospora crassa but little cross-reactivity to antisera raised against some beef heart complex I subunits. Plant complex I contains eight mitochondrial encoded subunits with the remainder being nuclear-encoded. Two of these mitochondrial-encoded subunits, nad7 and nad9, show homology to corresponding nuclear-encoded subunits inNeurospora crassa (49 and 30 kDa, respectively) and beef heart CI (49 and 31 kDa, respectively), suggesting a marked difference between the assembly of CI from plants and the fungal and mammalian enzymes. As well as complex I, plant mitochondria contain several type-II NAD(P)H dehydrogenases which mediate rotenone-insensitive oxidation of cytosolic and matrix NADH. We have isolated three of these dehydrogenases from beetroot mitochondria which are similar to enzymes isolated from potato mitochondria. Two of these enzymes are single polypeptides (32 and 55 kDa) and appear similar to those found in maize mitochondria, which have been localized to the outside of the inner membrane. The third enzyme appears to be a dimer comprised of two identical 43-kDa subunits. It is this enzyme that we believe contributes to rotenone-insensitive oxidation of matrix NADH. In addition to this type-II dehydrogenases, several observations suggest the presence of a smaller form of CI present in plant mitochondria which is insensitive to rotenone inhibition. We propose that this represents the peripheral arm of CI in plant mitochondria and may participate in nonphosphorylating matrix NADH oxidation.     

Genomic organization and evolution of the soybean SB92 satellite sequence

Repetitive DNA sequences comprise a large percentage of plant genomes, and their characterization provides information about both species and genome evolution. We have isolated a recombinant clone containing a highly repeated DNA element (SB92) that is homologous to ca. 0.9% of the soybean genome or about 10⁵ copies. This repeated sequence is tandemly arranged and is found in four or five major genomic locations. FISH analysis of metaphase chromosomes suggests that two of these locations are centromeric. We have determined the sequence of two cloned repeats and performed genomic sequencing to obtain a consensus sequence. The consensus repeat size was 92 bp and exhibited an average of 10% nucleotide substitution relative to the two cloned repeats. This high level of sequence diversity suggests an ancient origin but is inconsistent with the limited phylogenetic distribution of SB92, which is found an high copy number only in the annual soybeans. It therefore seems likely that this sequence is undergoing very rapid evolution.     

Ragweed sensitization alters pulmonary vascular responses to bronchoprovocation in beagle dogs

Theodorou, Andreas, Natalie Weger, Kathleen Kunke, KyooRhee, David Bice, Bruce Muggenberg, and Richard Lemen. Ragweed sensitization alters pulmonary vascular responses to bronchoprovocation in beagle dogs. J. Appl. Physiol.83(3): 912-917, 1997.In ragweed (RW)-sensitized beagle dogs, wetested the hypothesis that reactivity of the pulmonary vasculature wasenhanced with aerosolized histamine (Hist) and RW. Seven dogs wereneonatally sensitized with repeated intraperitoneal RW injections, and12 dogs were controls (Con). The dogs were anesthetizedwith intravenous chloralose, mechanically ventilated, and instrumentedwith femoral arterial and pulmonary artery catheters. Specific lungcompliance(CL_sp),specific lung conductance (G_sp),systemic vascular resistance index, and pulmonary vascular resistanceindex (PVRI) were measured before and after bronchoprovocation withHist and RW. After Hist inhalation (5 breaths of 30 mg/ml), both Conand RW dogs had significant (P < 0.05) decreases inCL_sp(51 ± 4 and 53 ± 5%, respectively) andG_sp (65 ± 5 and69 ± 3%, respectively), but only RW-sensitized dogs had asignificant increase in PVRI (38 ± 10%). After RW inhalation (60 breaths of 0.8 mg/ml), only RW-sensitized dogs had significant increases (62 ± 20%) in PVRI and decreases inG_sp (77 ± 4%) and CL_sp(65 ± 7%). We conclude that, compared with Con,RW-sensitized beagle dogs have increased pulmonary vasoconstrictiveresponses with Hist or RW inhalation.

     

Lack of coordination between glucose-6-phosphatase and gamma glutamyltranspeptidase activities in rat hepatocytes maintained in primary culture

Summary Glucose-6-phosphatase activity decreases whereas gamma glutamyltranspeptidase activity increases during hepatocarcinogenesis and the maintenance of hepatocytes in primary culture. This report describes the effect of culture conditions that are known to preserve hepatic glucose-6-phosphatase activity on gamma glutamyltranspeptidase activity. The results indicate that the regulation of glucose-6-phosphatase and gamma glutamyltranspeptidase activities is not coordinated in primary cultures of hepatocytes. This work was supported by a grant from the USPHS, NIH grant AG00439 awarded to Dr. Christopher C. Widnell and a Category I Research Development Award from the University of Pittsburgh to Dr. Kathleen Dobrosielski-Vergona. Editor's Statement Information communicated in this article contributes to a greater understanding of the mechanisms regulating liver cell metabolism and provides some further insight concerning the complexity of the controls involved in vitro, and presumably in vivo. David W. Barnes     

Serum indole-3-acetic acid in control subjects and newly abstinent alcoholics after an oral loading with L-tryptophan: a preliminary study using liquid chromatography with amperometric detection

Described in this paper is a rapid, isocratic assay for serum indole-3-acetic acid (IAA). The sample preparation involves only protein precipitation using sulfosalicylic acid, and the sensitivity of amperometric detection is in the picogram range. The chromatographic analysis time is approximately 4 min. The devised method was used for a longitudinal study of IAA levels in serum samples from control subjects and newly abstinent alcoholics. Dietary variations were eliminated by administering a 2.0-g loading dose of L-Trp to all subjects investigated. The results are presented in the form of cumulative frequency polygons. Preliminary data indicate no differences in IAA levels between newly abstinent alcoholics and control subjects.     

Ultrastructural changes in major organelles during spermatial differentiation inBangia (Rhodophyta)

Summary The major organelles within the cells of maleBangia atropurpurea (Roth) C. Ag. filaments undergo a series of ultrastructural transformations during the production of spermatia. Initially, thylakoids within the large axial chloroplast develop a reticulate pattern commencing at the central pyrenoid region. Subsequent changes involve loss of lobes and diminution of volume through division; chloroplasts in final stages contain a few dilated, distorted thylakoids and many plastoglobuli. During differentiation the large nucleolus disappears from the nucleus and four masses of chromatin aggregate near the nuclear envelope. Furrows originating from the nuclear envelope form double membranes around each of the chromatin masses and most of the nucleoplasm is eliminated. Several types of fibrillar vesicles are formed during the process and large floridean starch reserves are utilized. Multilamellar bodies and microbody-like structures occur within the cells during certain phases of spermatiogenesis.     

Applications of biofeedback to the treatment of asthma: A critical review

Both muscular and respiratory biofeedback procedures have been employed in attempts to reduce symptoms of bronchial asthma. Research relating to these approaches is reviewed in the present article. Biofeedback training both for facial muscle relaxation and for respiratory resistance decrease improves short-term pulmonary function in asthmatic individuals. These forms of training represent promising avenues for the management of asthma. However, unqualified endorsement of these procedures is premature, at the present time, since their influence on asthma-related variables other than pulmonary function has not been determined and since their long-term effects have not been investigated.     

Adherent cells in tumor immunity

The present studies explored the role of adherent cells in tumor immunity. Lymph node cells from mice bearing large tumors appeared to be maximally stimulated in vivo and incapable of further stimulation by cells of the same tumor in vitro. Removal of the adherent cell population resulted in a marked decrease in the spontaneous background activity of the remaining nonadherent cells and allowed these cells to undergo stimulation when cultured in the presence of mitomycin-blocked tumor cells. The role of the adherent cell in the maintenance of a state of continuous stimulation was further elucidated by experiments in which lymph node cell populations were reconstituted from the adherent and nonadherent subpopulations. It was also shown that adherent lymphoid cells from tumor-bearing mice, but not from normal mice, were capable of stimulating tumor-immune lymphocytes in a manner similar to intact mitomycin-blocked tumor cells.