Characterization of the recombinant Clostridium pasteurianum ferredoxin and comparison of its properties with those of the native protein

Ferredoxins (Fds) constitute an important class of nonheme iron-sulfur proteins. One of the most studied Fds is the [8Fe-8S] Fd from Clostridium pasteurianum. The gene for this Fd has previously been cloned and sequenced. We report the expression of this Fd in Escherichia coli, and the characterization and comparison of this recombinant protein to the native Fd. We have found that the purified recombinant protein has the same enzymatic, redox, magnetic and electronic properties as the native Fd isolated from C. pasteurianum, which indicates that the two [4Fe-4S] clusters present in the Fd were correctly formed in E. coli.     

Antibodies which recognize the C-terminus of the inhibitory guanine-nucleotide-binding protein (Gi) demonstrate that opioid peptides and foetal-calf serum stimulate the high-affinity GTPase activity of two separate pertussis-toxin substrates.

We investigated the mechanisms of receptor-mediated stimulation of high-affinity GTPase activity in response to opioid peptides and to foetal-calf serum in membranes of the neuroblastoma X glioma hybrid cell line NG108-15. Increases in GTPase activity in response to both of these ligands was abolished by prior exposure of the cells to pertussis toxin. Pertussis toxin in the presence of [32P]NAD+ catalysed incorporation of radioactivity into a broad band of approx. 40 kDa in membranes prepared from untreated, but not from pertussis-toxin-pretreated, cells. Additivity studies indicated that the responses to opioid peptides and to foetal-calf serum were mediated by separate guanine-nucleotide-binding proteins (G-proteins). Whereas opioid peptides produced an inhibition of adenylate cyclase in membranes of untreated cells, foetal-calf serum did not. Affinity-purified antibodies which recognize the C-terminus of the inhibitory G-protein identified a 40 kDa polypeptide in membranes of NG108-15 cells. These antibodies attenuated opioid-stimulated high-affinity GTPase activity, but did not markedly affect the response to foetal-calf serum. We conclude that receptors for the opioid peptides function via the inhibitory G-protein (Gi), whereas foetal-calf serum activates a second pertussis-toxin-sensitive G-protein, which has a C-terminal sequence significantly different from that of Gi.     

In-vitro and in-vivo responsiveness of the corpus luteum of the mare to gonadotrophin stimulation

Dispersed horse luteal cells were used to evaluate the ability of horse LH, hCG and PMSG to stimulate progesterone secretion in vitro. Morphological characterization of these cells before gonadotrophin stimulation indicated the presence of two populations of cells based on cell diameters. In luteal cells incubated as suspended cells, horse LH and hCG stimulated (P less than or equal to 0.05) progesterone production at all levels of treatment. Stimulation of progesterone secretion by hCG was greater (P less than or equal to 0.05) than by horse LH over the range of concentrations utilized. When mares (N = 7) received an intramuscular injection of 1000 i.u. hCG on Days 3, 4 and 5 after the end of oestrus, there was an increase (P less than or equal to 0.05), in peripheral progesterone concentrations beginning on Day 7 and continuing until Day 14 compared with controls (N = 7). Peripheral progesterone concentrations continued to be elevated in hCG-treated mares for Days 15-30 after oestrus in those mares that conceived. Although treatment with hCG increased progesterone concentrations, it had no influence on anterior pituitary release of LH as measured by frequency and amplitude of LH discharge. We conclude that the mare corpus luteum is responsive to gonadotrophins in vitro and that exogenous hCG can enhance serum progesterone concentrations throughout the oestrous cycle and early pregnancy.     

Effect of platelet-activating factor on the release of PGF-2 alpha and PGE-2 by separated cells of human endometrium

Synthetic platelet-activating factor (PAF-acether, 1-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine) caused a dose-dependent increase in the synthesis of prostaglandin (PG) E-2 by an enriched glandular, but not stromal, fraction of human endometrium removed in the secretory phase of the menstrual cycle. PAF-acether did not alter the synthesis of PGF-2 alpha by this fraction and consequently there was a significant reduction in the ratio of the amounts of PGF-2 alpha/PGE-2 released by the cells into the culture medium. The preimplantation human embryo secretes PAF, and these findings may have relevance to the local maternal recognition of pregnancy.     

Role of angiotensin II in renal hemodynamic functions during the initial stages of acute ureteral obstruction

This investigation examines the role of Angiotensin II in renal hemodynamic functions during acute unilateral ureteral obstruction (UUO) in a dog model. An electro magnetic flow probe was utilized to assess renal blood flow while the arteriovenous extraction technique of technetium 99m DTPA was utilized for the assessment of changes in filtration fraction and glomerular filtration rate. The effects of Angiotensin II receptor blockade on renal hemodynamic functions during acute UUO was evaluated in six dogs and compared to acute ureteral obstruction without receptor blockade in seven dogs. Angiotensin II blockade with (Sar1, Thr8)-Angiotensin II during UUO led to a striking increase in renal blood flow that was significantly different in comparison to normalized values from UUO alone (+delta 63 +/- 17 vs. +delta 22 +/- 6% at 30 min; p less than 0.05). There were, however, no significant differences in the magnitude of the decrease in filtration fraction and glomerular filtration rate in comparison to UUO alone. This investigation demonstrates that Angiotensin II has an inhibitory effect on the initial increase in renal blood flow with acute UUO. The possibility of successful pharmacologic intervention in the setting of UUO can be examined using animal models similar to the one described here. Pharmacologic treatment in the setting of acute UUO in patients might permit better preservation of renal function.     

Photoperiodic and genetic control of carbon partitioning in peas and its relationship to apical senescence

Apical senescence but not flower initiation is delayed by short days (SD) compared to long days (LD) in pea plants (Pisum sativum L.) of genotype E Sn Hr. We recently reported that delay of senescence correlated with slower reproductive development, suggesting that fruits are weaker sinks for assimilates under delayed senescence conditions. Thus, we have examined assimilate partitioning in peas to determine if genotype and photoperiod regulate relative sink strength. Assimilate diversion by developing fruit has been implicated in senescence induction. A greater percentage of leaf-exported ¹⁴C was transported to fruits and a smaller percentage to the apical bud of G2 peas (genotype E Sn Hr) in LD than in SD. Relatively more of the ¹⁴C delivered to the apical bud of G2 peas was transported to flower buds than to young leaves in LD as compared to SD. There was no striking photoperiodic difference in carbon partitioning in genetic lines without the Sn Hr allele combination. The Sn Hr allele combination and photoperiod may regulate the relative strength of reproductive and vegetative sinks. Photoperiodic differences in sink strength early in reproduction suggest that these genes regulate sink strength by affecting the physiology of the whole plant. High vegetative sink strength in SD may maintain assimilate supply to the apical bud, delaying senescence.     

Characterization of a renal tubular epithelial cell line which secretes the autologous target antigen of autoimmune experimental interstitial nephritis

Proximal tubular epithelial cells from mice which develop autoimmune interstitial nephritis were found to express the nephritogenic target antigen, 3M-1. Anti-3M-1 mAbs (alpha 3M-1-Ab) were used to positively select for 3M-1-secreting tubular epithelium and, after stabilization in culture, this new cell line (MCT) was examined for the production of several moieties important to either immune interactions or to the development of extracellular matrix. Alkaline phosphatase-staining MCT cells also express epithelial growth factor receptors with a Kd of 0.87 nM and an epithelial growth factor receptor constant (Ro) of 2.1 X 10(4) receptors/cell. MCT culture supernatants contain greater amounts of laminin, and types IV and V procollagens compared to types I and III procollagens, and growing MCT cells on type I collagen matrix causes them to preferentially secrete even more type IV and V procollagen. The 30,000-Mr 3M-1 antigen could be immunoprecipitated from biosynthetically labeled MCT cell supernatants with alpha 3M-1-Ab. An identical-sized moiety was isolated by immunoaffinity chromatography from collagenase-solubilized mouse kidney tubular basement membranes. The 3M-1 antigen can be found on the MCT cell surface by radioimmunoassay, or deposited in a linear array in the extracellular matrix surrounding the MCT cells in culture by immunofluorescence. Mature messenger RNA species for both class I and class II major histocompatibility complex (MHC) molecules were detected by Northern hybridization, and their corresponding cell surface gene products were detected by cytofluorography of MCT cells stained with haplotype-specific antibodies. Both the cell surface 3M-1 and the small amounts of detected class II MHC molecules appear to be biologically functional, as MCT cells can support the proliferation of 3M-1-specific, class II MHC-restricted helper T cells in culture. These findings suggest that MCT cells provide all the necessary biological parameters for interfacing both as the target of a nephritogenic immune response, and as a potential source for new extracellular matrix which develops as a fibrogenic response to interstitial nephritis.     

Genome instability in interspecific cell hybrids I. Unstable expression of genes in divergent cell hybrids

Somatic cell hybrids between cells of widely divergent mammalian species display a range of chromosomal and genetic anomalies which may be the equivalent of the “genomic shock” phenomena observed in many plant and animal interspecific hybrids. Mouse-kangaroo hybrids show extreme segregation and fragmentation of the kangaroo chromosomes. Here 1 show that, in addition to the chromosomal instability, some hybrids display unstable expression of three genes borne on the kangaroo active maternal X chromosome. These genes (HPRT, G6PD andPGK) may be co-ordinately inactivated at high frequency, then reactivated once more. I suggest that this reversible inactivation in interspecific hybrids may be the result of an unstable change at an X inactivation centre located in the kangaroo X_q.     

Pleural and extrapleural interstitial liquid pressure measured by cannulas and micropipettes

In 15 anesthetized apneic, oxygenated rabbits we simultaneously measured pleural liquid and interstitial extrapleural parietal pressures by using catheters and/or cannulas and micropipettes connected to a servonull system. With the animal in lateral posture, at an average recording height of 4.4 +/- 0.9 (SD) cm from the most dependent part of the cavity, the extrapleural catheter and the pleural cannula yielded -2.5 +/- 0.6 and -5.5 +/- 0.2 cmH2O; the corresponding values for micropipette readings in the two compartments were -2.4 +/- 0.6 and -5.4 +/- 0.4 cmH2O, respectively (not significantly different from those measured with catheters and cannulas). In the supine animal, interstitial extrapleural catheter pressure data obtained at recording heights ranging from 15 to 80% of pleural cavity lay on the identity line when plotted vs. the micropipette pressure values simultaneously gathered from the same tissues. We conclude that 1) micropipettes and catheters-cannulas yield similar results when recording from the same compartment and 2) the hydraulic pressure in the parietal extrapleural interstitium is less negative than that in the pleural space.