Cellular and developmental patterns of expression of Ret and glial cell line-derived neurotrophic factor receptor alpha mRNAs

 Glial cell line-derived neurotrophic factor (GDNF) has recently been shown to signal by binding to GDNF receptor-alpha (GDNFR-α), after which the GDNF-GDNFR-α associates with and activates the tyrosine kinase receptor Ret. We have localized Ret messenger RNA (mRNA) in the developing and adult rodent and compared with to the expression of GDNF and GDNFR-α mRNA. Ret mRNA is strongly expressed in dopamine neurons and α-motorneurons as well as in thalamus, ruber and occlumotor nuclei, the habenular complex, septum, cerebellum, and brain stem nuclei. Ret mRNA was also found in several sensory systems, in ganglia, and in nonneuronal tissues such as teeth and vibrissae. Very strong Ret mRNA signals are present in kidney and the gastrointestinal tract, where Ret and GDNF mRNA expression patterns are precisely complementary. The presence of Ret protein was confirmed in adult dopamine neurons using immunohistochemistry. GDNFR-α mRNA was strongly expressed in the developing and adult dopamine neurons. It was also found in neurons in deep layers of cortex cerebri, in hippocampus, septum, the dentate gyrus, tectum, and the developing spinal cord. In the kidney and the gastrointestinal tract, GDNFR-α mRNA and Ret mRNA distribution overlapped. Dorsal root ganglia, cranial ganglia, and developing peripheral nerves were also positive. GDNFR-α was additionally found in sensory areas and in developing teeth. Sensory areas included inner ear, eye, olfactory epithelium, and the vomeronasal organ, as well as developing tongue papillae. The temporospatial pattern of expression of GDNFR-α mRNA did not always match that of Ret mRNA. For instance, GDNFR-α mRNA was also found in the developing ventral striatum, including the olfactory tubercle, and in hippocampus. These areas seemed devoid of Ret mRNA, suggesting that GDNFR-α might also have functions unrelated to Ret. Received: 2 January 1997 / Accepted: 26 February 1997     

Survival outcomes of patients with giant cell myocarditis bridged by ventricular assist devices

Giant cell myocarditis is a highly lethal disorder characterized by rapidly progressive congestive heart failure. The aim of this study was to describe the clinical course of patients with giant cell myocarditis who received a ventricular assist device. Patients with giant cell myocarditis were identified from the Multicenter Giant cell Myocarditis Registry. Bridging to cardiac transplantation in the giant cell myocarditis patients who received a ventricular assist device was compared with bridging in the general population of heart failure patients, as reported in the literature. Median posttransplantation survival for patients with giant cell myocarditis who received and did not receive ventricular assist devices was calculated by the Kaplan-Meier method and compared with use of the log-rank test. Nine patients with giant cell myocarditis who received ventricular assist devices were identified. Seven patients survived to transplantation, four were alive 30 days posttransplantation, and two survived to 1 year. The rate of successful bridging to transplantation in seven of nine patients (78%) is similar to that reported for other ventricular assist device recipients. Posttransplantation survival of 57% (4 of 7) at 30 days and 29% (2 of 7) at 1 year was significantly lower compared with 93% 1-year survival of the 30 patients with giant cell myocarditis who did not receive ventricular assist devices before transplantation (p<0.001). Ventricular assist devices can be an effective bridge to transplantation for patients with heart failure caused by giant cell myocarditis. Although their posttransplantation survival was poor in our series, a few patients had long-term survival.     

Localization of actin in mammalian spermatozoa: a comparison of eight species

The distribution of monomeric and polymeric actin in spermatozoa from the bull, boar, rabbit, human, rat, mouse, golden hamster, and guinea pig has been examined by using a monoclonal antiactin antibody and NBD-phallacidin. Actin was present in sperm from each species. When the monoclonal antibody was used, there was a species-specific distribution and intensity of fluorescence, but no generalized pattern. Specific fluorescence was noted in the neck and principal piece of human sperm; in the postacrosomal region, neck, and midpiece of bull and boar sperm; in the postacrosomal region, neck, and principal/equatorial segment border of rabbit sperm; in the neck region of hamster sperm; and in the neck, midpiece, and principal piece of rat, mouse, and guinea pig sperm. Sperm from all eight species displayed no specific fluorescence with NBD-phallacidin, indicating that actin was present in a nonfilamentous form. SDS extracts of sperm were analyzed by SDS-PAGE and Western blotting; in sperm from each species, a 42-kD protein with specific affinity for the monoclonal antibody was present.     

The slide centrifuge gram stain as a urine screening method.

A slide centrifuge Gram stain procedure was performed to screen for bacteriuria 4161 urine specimens submitted in urine preservative tubes for routine culture. For slide centrifuge Gram staining, each urine sample was mixed well. Thereafter, 0.2 mL of each sample was placed, using a pipette, into a slide centrifuge chamber and centrifuged at 2,000 rpm for 5 minutes. The slides were heat fixed, Gram stained, and read by laboratory personnel who scanned 12 consecutive oil-immersion fields using a set pattern. The presence of the same organism in six or more fields was defined as a positive urine screen. Urine samples were cultured using a 0.001-mL loop and a comparison of culture growth with slide centrifuge screening was made. When growth of 100,000 or more colony-forming units per milliliter (CFU/mL) was the reference for comparison, the screen had a sensitivity rate of 98%, a specificity rate of 90%, a negative predictive value of 99%, and a positive predictive value of 65%. When a lower colony count of 10,000 or more CFU/mL was the reference for comparison, the screen had a sensitivity rate of 88%, a specificity rate of 95%, a negative predictive value of 96%, and a positive predictive value of 84%. The slide centrifuge Gram stain is a very sensitive screening method to detect bacteriuria in an adult male population.     

Hoechst staining and exposure to UV laser during flow cytometric sorting does not affect the frequency of detected endogenous DNA nicks in abnormal and normal human spermatozoa

Controlling the sex of offspring by the separation of X and Y chromosome-bearing spermatozoa using flow cytometry has been reported as a clinical technique aiding prevention of X-linked diseases. Although this technique has resulted in several hundred normal births in animals and at least one human birth, there is still concern over its genetic safety due to the involvement of two potentially mutagenic agents: UV light and the fluorochrome dye, Hoechst 33342 (H33342). Human spermatozoa, particularly those considered abnormal, may be more likely to suffer DNA damage following exposure to mutagenic agents, compared with other mammalian species. The stability of normal fresh and decondensed human spermatozoa were examined after exposure to a range of levels of UV and H33342 staining, using an assay that detects endogenous nicks in the DNA of spermatozoa. The stability of abnormal and normal, fresh and frozen-thawed human spermatozoa was examined following UV laser, H33342 staining and flow cytometry treatments utilizing the same assay. There was an increase in the presence of endogenous nicks when spermatozoa were decondensed compared with fresh spermatozoa. There was no increase in the incidence of nicks in any group of spermatozoa after UV and fluorochrome exposure compared with controls without exposure.      

Modulation of disease severity of dystrophic epidermolysis bullosa by a splice site mutation in combination with a missense mutation in the COL7A1 gene

Dystrophic epidermolysis bullosa (EBD) is a clinically heterogeneous skin disorder, characterized by abnormal anchoring fibrils (AF) and loss of dermal-epidermal adherence. EBD has been linked to the COL7A1 gene at chromosome 3p21 which encodes collagen VII, the major component of the AF. Here we investigated two unrelated EBD families with different clinical phenotypes and novel combinations of recessive and dominant COL7A1 mutations. Both families shared the same recessive heterozygous 14 bp deletion at the exon-intron 115 boundary of the COL7A1 gene. The deletion caused in-frame skipping of exon 115 and the elimination of 29 amino acid residues from the pro-alpha1(VII) polypeptide chain. As a result, procollagen VII was not converted to collagen VII and the C-terminal NC-2 propeptide which is normally removed from the procollagen VII prior to formation of the anchoring fibrils was retained in the skin. All affected individuals also carried missense mutations in exon 73 of COL7A1 which lead to different glycine- to-arginine substitutions in the triple-helical domain of collagen VII. Combination of the deletion mutation with a G2009R substitution resulted in a mild phenotype. In contrast, combination of the deletion with a G2043R substitution led to a severe phenotype. The G2043R substitution was a de novo mutation which alone caused a mild phenotype. Thus, different combinations of dominant and recessive COL7A1 mutations can modulate disease activity of EBD and alter the clinical presentation of the patients.      

Evidence for platelet-activating factor as a late-phase mediator of chronic pancreatitis in the rat.

The role of platelet-activating factor (PAF) as a mediator of pancreatic inflammation was examined in the rat pancreatic duct ligation model of obstructive pancreatitis. Pancreatic generation of PAF, as measured by bioassay (ie, platelet [3H]serotonin secretion), was determined at various times after induction of inflammation. Tissue levels of PAF in the normal pancreas averaged 600 +/- 49 pg/g, but PAF was not detectable during the initial 24 hours of pancreatitis, a time when the inflammatory reaction would be considered acute, that is, during the period of maximal serum amylase release and the development of interstitial edema. However a substantial increase in pancreatic PAF levels (12 times control levels) was observed 7 to 14 days after duct ligation during the late-phase response interval similar to the situation characteristic of chronic pancreatitis in which parenchymal atrophy, fibrosis, and pancreatic insufficiency evolve. One week after duct ligation when PAF levels peaked, an evaluation was made of the effects of PAF antagonists (BN52021 and WEB2170) on pancreatic lesions using Evan's blue extravasation, pancreatic myeloperoxidase (MPO) activity, and acid phosphatase activity in peritoneal lavage fluid. BN52021 or WEB2170 treatment was shown to reduce pancreatic damage and inflammation significantly. Long-term in vivo administration of exogenous PAF (20 micrograms/kg/hr for 7 days) exhibited a reduction of [3H]thymidine uptake into and amylase release from pancreatic acini in vitro. Our observations 1) that pancreatic PAF levels increased significantly during the chronic phase of obstructive pancreatitis induced by duct ligation; 2) that inhibition of the action of PAF, through specific receptor antagonism, caused an attenuation of pancreatic lesions; and 3) that chronic administration of PAF resulted in decreased pancreatic regeneration and exocrine function are consistent with a pivotal role for PAF as a late-phase inflammatory mediator in chronic pancreatitis in rats.     

Intraocular grafting of cultured brain tissue: growth, vascularization and neuron survival in locus coeruleus and cortex cerebri

Locus coeruleus and cortex cerebri from embryonic (ED 17) and newborn rats were kept 4 days in tissue culture under conditions maintaining organotypic features and then transplanted to the anterior chamber of the eye of adult rats. Vascularization from the host iris was delayed in pre-cultured grafts as compared to directly grafted material. In spite of this, several morphological parameters developed normally. Thus, pre-cultured grafts grew considerably in oculo. Falck-Hillarp histochemistry showed that grafts of cortex cerebri received an adrenergic innervation from the host iris and that locus coeruleus grafts contained central adrenergic neurons capable of innervating a sympathetically denervated host iris. The successful combination of tissue culture and intraocular transplantation should permit the selective advantages of both techniques to be applied to the same tissue pieces, generating new information unobtainable by either method alone.