Search

Abstract We have previously shown that the partial disruption of the gene for atrial natriuretic peptide (ANP) results in a salt-sensitive phenotype. The present study examined the possibility that alterations in either the ANP natriuretic pathway or endothelin (ET) system in the kidney of the salt-challenged ANP +/− mouse was responsible for its salt-sensitive phenotype. Plasma ANP levels and renal cGMP activity were increased in response to a salt load in both ANP +/+ and +/− mice. However, the mRNA expression of proANP was found to be increased only in the ANP +/− kidney along with its guanylyl cyclase-linked receptor, NPRA; the upregulation of NPRA mRNA was limited to the renal medulla. This suggests that the renal ANP pathway remains capable of responding to a salt load in the ANP +/− animal, but may be compensating for other dysfunctional pathways. We also report a significant increase in renal ET-1 mRNA and ETA receptor protein expression in medulla and cortex of the salt-treated, ANP +/− mouse, but not its wild-type counterpart. In fact, ETA expression decreased in the renal cortex of the ANP +/+ salt-treated animal. The ETB receptor expression was not affected by diet in either genotype. We hypothesize that the salt-sensitive hypertension in the ANP +/− mouse is exacerbated, and possibly driven by the vasoconstrictive effects resulting from an upregulated ET-1/ETA pathway.

Using magnetic resonance imaging (MRI), the internal neural and craniofacial malformations of a cyclopic fetus are described. External facial features were characterized by a tubular proboscis situated above a single eye slit. The brain was recognized as ‘pancake’ type alobar holoprosencephaly (a condition where the undifferentiated telencephalon partially surrounds a monoventricle). Displacement of some bones that normally contribute to the orbit could be clearly discerned. Absence of neural structures (e.g. falx cerebri, corpus callosum) and missing components of the ethmoid bone indicated a midline deficit. This correlates with proposed theories of cyclopic embryopathy, which suggest that the prechordal plate and the neural crest cells are affected during the third week of gestation in cyclopia.

Multiple families of cyclic nucleotide phosphodiesterases (PDE) have been described, and the regulated expression of these genes in cells is complex. Although cAMP is known to control the expression of certain PDE in cells, presumably reflecting a system of feedback on cAMP signaling, relatively little is known about the influence of non-cAMP signaling systems on PDE expression. In this study, we describe a novel mechanism by which activators of the protein kinase C (PKC)-Raf-MEK-ERK cascade regulate phosphodiesterase 4D (PDE4D) expression in vascular smooth muscle cells (VSMC) and assess the functional consequences of this effect. Whereas a prolonged elevation of cAMP in VSMC resulted in a protein kinase A (PKA)-dependent induction of expression of two PDE4D variants (PDE4D1 and PDE4D2), simultaneous activation of both the cAMP-PKA and PKC-Raf-MEK-ERK signaling cascades blunted this cAMP-mediated increase in PDE4D expression. By using biochemical, molecular biological, and pharmacological approaches, we demonstrate that this PDE4D-selective effect of activators of the PKC-Raf-MEK-ERK cascade was mediated through a mechanism involving altered PDE4D mRNA stability and markedly attenuated the cAMP-mediated desensitization that results from prolonged activation of the cAMP signaling system in cells. The data are presented in the context of activators of the PKC-Raf-MEK-ERK cascade having both short and long term effects on PDE4D activity and expression in cells that may influence cAMP signaling.

This study was undertaken to examine ultrastructural changes induced by dopamine in fish prolactin cells. Tilapia adenohypophyses were incubated with dopamine and evaluated by electron microscopy. The quantities of rough endoplasmic reticulum (RER) in prolactin cells increased and the number of secretory granules were decreased by dopamine (10⁶ mol/l) treatment. Another set of adenohypophysial tissues was placed back into control medium for 10 min following a 3 h incubation period with dopamine (10⁶ mol/l) (RE10 min group). This group had significantly less RER than the 3 h dopamine-treated tissue, and the shape of many granules in the RE10 min group changed from spherical to rod-like. In addition, some of the granule content appeared to diffuse out of granules since some were not fully surrounded by membrane. It was therefore hypothesized that the rod-shaped granules might be the result of prolactin secretion by diffusion.

The parameter of sperm viability in hypoosmotic solution (VHOS) provides information concerning the membrane integrity of the sperm head. The objective of this study was to determine the association between the VHOS parameter and the sperm penetration assay. The VHOS parameter correctly predicted 70.0-71.4% of the failed sperm penetration assay samples in the short duration preincubation groups. A combination of both the VHOS parameter and the hypoosomotic sperm swelling (HOS) test significantly reduced the number of false negative results. In general, a sperm sample with an abnormal VHOS result and an abnormal HOS test result would be associated with a negative sperm penetration assay. Washing by centrifugation appeared to weaken the sperm head membranes while the swim-up method selected for sperm with strong head and tail membranes. After the various processing methods unique changes in the integrity of the sperm head and tail membranes for each sperm sample may help to identify the optimal method of preparation for individual patients undergoing the newer assisted reproductive technologies such as sperm microinjection.

Recently, there have been concerns regarding the presence of reactive oxygen species (ROS) produced during sperm processing for insemination. However, the sperm wash methods that yielded low ROS levels also had low sperm recovery after processing. The objective of this study was to compare sperm recovery after swim-up from pellet, overlay, and 2-layer Percoll wash methods with the recovery after the double method wash. The latter method consisted of a combination of 2 sperm wash methods, namely, the overlay and the Percoll method. Motile sperm were first collected through the overlay method. The leftover semen was then processed through the 2-layer Percoll method to scavenge motile sperm and the resultant pellet combined with the pellet from the overlay method. In this manner, the level of ROS was kept to a minimal, sperm recovery was improved, and a mixture of sperm with different surface properties was produced as a result of using different processing methods. The results indicated an improvement in sperm recovery and in total sperm motility in noncryopreserved sperm after using the double method wash when compared with the other wash methods. The study suggests that the double method wash is a feasible method for processing sperm for insemination.

The protocol for intrauterine insemination (IUI) involves sperm processing using different methods that have produced varying results. These sperm wash methods do not take into consideration the problems of the exact timing of ovulation and the requirements of sperm cells at different stages of capacitation. The objectives of this study were 1) to use the double method wash previously reported to produce a mixture of different populations of sperm cells and determine the pregnancy outcome after IUI and 2) to compare the sperm kinematic parameters after the double method wash with those after the centrifuge (or whole-population) wash method. Patients were divided into either the double method group (n = 119) or the centrifuge method group (n = 76). The Hamilton-Thorn HTM-C automated sperm motility analyzer (Hamilton-Thorn Research, Danvers, MA) was used to analyze sperm motility parameters. Pregnancy outcomes were evaluated after controlled ovarian stimulation and IUI. An almost 2-fold increase was seen in the pregnancy rate with the double method wash compared with the centrifuge method wash. Sperm motility and velocity were also enhanced in the double method wash groups. The results support the usefulness of the double method wash for the preparation of sperm for IUI.

Treatment of Chinese hamster ovary (CHO) cells by the aldehyde containing calpain inhibitor I resulted in the induction of a 35-kDa protein that was partially sequenced and shown to be a member of the aldo-keto reductase superfamily (Inoue, S., Sharma, R. C., Schimke, R. T., and Simoni, R. D. (1993) J. Biol. Chem. 268, 5894-5898). Using rapid amplification of cDNA ends polymerase chain reaction, we have sequenced the cDNA for this protein (CHO reductase). This enzyme is a new member of the aldo-keto reductase superfamily and shows greatest amino acid sequence identity to mouse fibroblast growth factor-regulated protein and mouse vas deferens protein (92 and 80% sequence identity, respectively). The enzyme exhibits about 70% sequence identity with the aldose reductases (ALR2; EC 1.1.1.21) and about 47% with the aldehyde reductases (ALR1; EC 1.1.1.2). Northern analysis showed that it is induced in preference to either ALR1 or ALR2 and RNase protection assays showed gene expression in bladder, testis, jejunum, and ovary in descending order of expression. The cDNA for this inducible reductase was cloned into the pET16b vector and expressed in BL21(DE3) cells. Expressed CHO reductase showed kinetic properties distinct from either ALR1 or ALR2 including the ability to metabolize ketones. This protein joins a growing number of inducible aldo-keto reductases that may play a role in cellular regulation and protection.

Rat BNP-45 is the main circulating form of BNP in rat plasma. To understand the role of BNP in physiological and pathophysiological conditions, a specific radioimmunoassay (RIA) for the quantitative determination of the peptide in plasma and tissues is necessary. An assay using rBNP-45 as the standard in conjunction with antisera directed against this peptide has not been described in the literature, though some investigators have reported values ranging from 0.73-2.0 pmol/L using either BNP-26 or BNP-32 as the standard peptide. Unfortunately, these forms of BNP do not exist in rat plasma. In our studies, we have developed a specific RIA for rBNP-45 using rBNP-45 as the standard peptide and Tyr0 rBNP-45 as the radioligand. We have used two specific antisera for assay purposes; one against rBNP-45, and the second to a peptide composed of the first 20 amino acids of rBNP-45 (rBNP[1-20]). The recovery of various amounts of rBNP-45 added to control plasma was 50-80% depending on the method of extraction and purification. The interassay and intraassay coefficients of variation were 12% and 6% respectively. Values obtained were similar for blood sampled by either cardiac puncture, decapitation, or aortic puncture. The method was used to measure rBNP-45 in the plasma of normal (WKY) and Spontaneously Hypertensive (SHR) rats. The values obtained were 5.46 /pm 0.43 and 19.6 /pm 2.36 pmol/L respectively. The rat atrial natriuretic peptide (ANP[99-126]) values in the same extracts were 23.2 /pm 0.45 and 51.6 /pm 3.16 pmol/L.

The induction of cyclic GMP formation in target tissues, i.e. vascular smooth muscle and endothelial cells, by atrial natriuretic factor is followed by its egression into plasma and urine. Since the extracellular appearance of this cyclic nucleotide is used as a marker of atrial natriuretic factor's biological activity, the present study was designed to investigate the characteristics of its egression to the extracellular fluid. In contrast to cyclic AMP, whose profile of egression in time closely follows its intracellular levels, cyclic GMP egression begins with the intracellular decline but continues for a prolonged period, even accumulating for up to 24 h in the extracellular medium. The relative egression of cyclic GMP decreases slightly in the presence of phosphodiesterase inhibitors. On the other hand, the process is sensitive to temperature, inhibited by such agents as probenecid, and occurs against a gradient of 7 orders of magnitude. Large increases of cyclic AMP, as induced by forskolin, can effectively compete for the cyclic GMP transport system, resulting in a 3-fold rise in intracellular cyclic GMP levels, which corresponds to a 3-fold decline of extracellular accumulation. Although the biological significance of cyclic GMP egression is unknown, the results of this study suggest that the process may be one of the significant contributors to the control of cyclic GMP levels in the cell with potential physiological consequences.

Dopamine is considered to be the major physiological tonic inhibitor of prolactin release, yet there is increasing evidence showing that it can also stimulate prolactin release from lactotrophs. In primary cultured lactotrophs, the major dopamine receptors responsible for inhibiting prolactin release are dopamine D2receptors. A dopamine receptor subtype may be responsible for the stimulatory action, yet one cannot exclude the possibility that a dopamine D2receptor can play dual roles. This study was therefore undertaken to investigate if dopamine both stimulates and inhibits prolactin secretion through activation of the same dopamine D2receptor. GH4ZR7cells, which have only one type of dopamine receptors—D2s, were perifused with different concentrations of dopamine, and the perifusate was assayed for prolactin; 10−7mol/L dopamine stimulated prolactin release (p<0.05;n=5), whereas 5×10−4mol/L dopamine inhibited prolactin secretion (p<0.05;n=5). In the pertussis toxin-treated cells, 10−7mol/L dopamine stimulated prolactin release (p<0.05;n=5), and 5×10−4mol/L dopamine did not significantly change the rate of prolactin release. These results indicate that both the stimulatory and inhibitory actions of dopamine are likely mediated by the same D2receptor subtype, since GH4ZR7cells express only D2sreceptors. They also confirm that the inhibitory action of dopamine is mediated through a Giprotein; and the stimulatory action of dopamine is mediated through a PTX-insensitive pathway. These findings suggest that D2receptors are coupled to both Giand Gsproteins.

AbstractBetween 1986 and 1989 three Vietnamese migrants presented at the Perth Chest Clinic with multiple‐drug resistant tuberculosis of the lungs. This resistance was to both rifampicin and isoniazid in addition to other antituberculous drugs. Although all three had a long history of past intermittent treatment for tuberculosis and extensive active disease, they were not detected in spite of what should have been adequate premigration screening. This report is 1o alert those medical personnel concerned with the process of migrant health screening and surveillance. The implications of such multi‐drug resistant disease on tuberculosis control in Australia is also briefly discussed. (Aust NZ J Med 1991; 21: 457–459.)