Cloning and characterization of bovine low molecular weight GTPases (Rac1 and Rac2) and rho GDP-dissociation inhibitor 2 (D4-GDI).

GTPases of the Rho family play important roles in human leukocyte signal transduction pathways; however, little is known about the function of these proteins in bovine cells. In the present studies, we isolated molecular clones of bovine Rac1, Rac2, and the Rac/Rho GTPase regulatory protein D4-GDP dissociation inhibitor (D4-GDI) from a bovine bone marrow cDNA library. These clones contained complete open reading frames, encoding 192, 192, and 200 amino acids, respectively. Comparison of the bovine amino acid sequences with those of other species demonstrated a high degree of identity of these proteins across all species, suggesting that these proteins likely play conserved functional roles in bovine leukocyte signal transduction pathways. Comparative Western blotting of these proteins in human and bovine neutrophil cytosol demonstrated that Rac2 was the predominant Rac species and that D4-GDI was the predominant GDI species in bovine neutrophil cytosol. Despite the high degree of homology between human and bovine Rac2, some of the anti-peptide antibody probes prepared against human Rac2 failed to recognize the bovine homologue. We also showed by subcellular fractionation techniques that Rac2 is localized primarily to the cytosolic compartment of resting bovine neutrophils, but is translocated to the plasma membrane after stimulation with PMA. These findings suggest that Rac2 does play a role in bovine neutrophil activation. In addition, these data will be helpful in developing more specific probes for investigating the role of these proteins in bovine leukocyte signal transduction pathways and for studying various inflammatory diseases in cattle.

Cloning and expression of bovine p47-phox and p67-phox: comparison with the human and murine homologs.

Neutrophils play an essential role in bovine cellular host defense, and compromised leukocyte function has been linked to the development of respiratory and mucosal infections. During the host defense process, neutrophils migrate into infected tissues where they become activated, resulting in the assembly of neutrophil membrane and cytosolic proteins to form a superoxide anion-generating complex known as the NADPH oxidase. Two of the essential cytosolic components of the NADPH oxidase are p47-phox and p67-phox. Currently, only the human and murine homologs of these proteins have been sequenced. Because of the important role neutrophils play in bovine host defense, we carried out studies to clone, sequence, and express bovine p47-phox and p67-phox. Using polymerase chain reaction (PCR) cloning techniques and a bovine bone marrow cDNA library, we have cloned both of these bovine NADPH oxidase cytosolic components. Comparison of the bovine sequences with those of the human and murine homologs showed that they were highly conserved, but also revealed important information regarding key structural features of p47-phox and p67-phox, including location of putative phosphorylation sites. Functional expression of bovine p47-phox and p67-phox showed that these proteins could substitute for the human proteins in reconstituting NADPH oxidase activity in a cell-free assay system, again demonstrating the high degree of conservation between human and bovine homologs. This study greatly contributes to our understanding of the potential structural/functional regions of p47-phox and p67-phox as well as providing information that can be used to study the role of neutrophils in bovine inflammatory diseases.

Lumbosacral anterolateral spinal arteries and brief review of "accessory" longitudinal arteries of the spinal cord.

The lumbosacral anterolateral spinal arteries (LALSA) were studied in 14 injected lumbosacral spinal cords. Contrary to many previously published opinions, which claimed that virtually all of the formerly described "accessory" longitudinal arteries of the vasa corona, were too inconstant to be of significance, the LALSA in this series were reliably present bilaterally and averaged a length of 8.4 cm. They ran in the acute angles formed by the emergence of the fascicles of the lumbosacral anterior spinal nerve roots and in this position, served as the origin for most of the proximal radicular arteries that supply the cranial half of the motor roots of the cauda equina. A review of the literature and analysis of other accessory longitudinal spinal arteries revealed that two other pairs of accessory spinal vessels, the lateral cervical spinal arteries (LCSA) and the lateral spinal arterial axes (LSA), have a demonstrable functional role and were sufficiently constant, along with the LALSA, to warrant inclusion in detailed descriptions of human spinal cord vasculature.

Platelet-activating factor induces a concentration-dependent spectrum of functional responses in bovine neutrophils.

We characterized the dose response of bovine neutrophils to platelet-activating factor (PAF) with respect to the following functions: calcium flux and membrane potential changes, actin polymerization, degranulation, and the production and/or priming of the oxidative burst. PAF at very low concentrations (10(-10) and 10(-9) M) caused changes in intracellular calcium and membrane potential in bovine neutrophils, whereas moderate PAF concentrations (> or = 10(-7) M) resulted in increased actin polymerization. Degranulation responses to PAF were more complex: low concentrations (10(-9) M) caused secretory granule degranulation, moderate doses (> or = 10(-7) M) caused specific granule degranulation, whereas azurophil degranulation only occurred at high (10(-5) M) PAF concentrations. Treatment of bovine neutrophils with PAF at concentrations > or = 10(-7) M also caused up-regulation of the adhesion molecules Mac-l and L-selectin. PAF stimulation resulted in a very weak [compared to phorbol myristate acetate (PMA)] oxidative burst in bovine neutrophils, and only at high (10(-6) M) concentrations. Unlike human neutrophils, bovine neutrophils were poorly primed by PAF treatment. Only high concentrations of PAF (10(-5) M) caused an increased rate of PMA-stimulated superoxide production, although lower doses of PAF did reduce the lag time preceding the PMA-induced oxidative burst. The overall pattern that can be inferred is that lower concentrations of PAF promote neutrophil sensitivity and interaction by selective degranulation, up-regulation of adhesion molecules, and increased actin polymerization. In contrast, higher PAF concentrations can promote, albeit weakly, more direct bactericidal responses, such as the release of reactive oxygen species and granule enzymes. The ability of PAF to modulate a graded response in bovine neutrophils would allow the cell to respond proportionally to the severity of a stimulus.

Cloning and sequencing of the bovine flavocytochrome b subunit proteins, gp91-phox and p22-phox: comparison with other known flavocytochrome b sequences.

Neutrophils play an essential role in the cellular defense of the bovine mammary gland and compromised leukocyte function has been linked to the development of bovine mastitis. During mastitis, large numbers of leukocytes migrate into the mammary tissues where they become activated, resulting in the assembly of neutrophil membrane and cytosolic proteins to form a superoxide anion-generating complex known as the NADPH oxidase. The key membrane-associated component of the NADPH oxidase is flavocytochrome b, which is a heterodimer of p22-phox and gp91-phox. Currently, only the human, porcine, murine, and rattus p22-phox and the human, porcine, and murine gp91-phox gene sequences are known. Because of the important role neutrophils play in bovine host defense, we carried out studies to clone, sequence, and analyze expression of bovine flavocytochrome b. Using polymerase chain reaction cloning techniques and a bovine spleen cDNA library we have cloned both of the bovine flavocytochrome b subunits, p22-phox and gp91-phox. Comparison of the bovine sequences with those of other species also revealed important information regarding key structural features of gp91-phox and p22-phox, including location of putative glycosylation sites. This study greatly contributes to our understanding of the potential functional sites of the flavocytochrome b subunits as well as providing information that can be used to study the role of neutrophils in bovine inflammatory diseases such as mastitis.

Formation of binucleated cardiac myocytes in rat heart: I. Role of actin-myosin contractile ring.

Cardiac myocytes in rat hearts lose their ability to undergo cytokinesis between day 3 and day 4, resulting in the formation of binucleated myocytes. Failure in the formation of the actin-myosin contractile ring could cause cardiac myocytes to be defective in cytokinesis. Enzymatically isolated cardiac myocytes from 2- and 4-day-old rats were employed to investigate the organisation and distribution of actin, myomesin, and myosin by rhodamine phalloidin, anti-myomesin, and isoform-specific anti-myosin antibodies, respectively. Interestingly, the actin-myosin contractile ring was formed in mitotic myocytes from both 2- and 4-day-old animals. The changes in organisation and distribution of actin, myosin and myomesin in mitotic myocytes from 4-day-old rats were similar to those from 2-day-old rats, except that there were longitudinal actin filaments in the cytoplasm of mitotic myocytes from 4-day-old rats. In mitotic myocytes from both 2- and 4-day-old rats, actin disassembled in prometaphase, concentrated in the equator of the mitotic spindle in late anaphase, and formed a circumferential intensely staining band in early telophase. Cytoplasmic myosin was evenly distributed in the cytoplasm as small spots, and appeared to associate with the cell membrane from interphase to early anaphase. It became progressively more concentrated in association with the cortical membrane in the equator region in late anaphase, formed a ring-like structure in early telophase, and remained associated with adjacent membrane at the cleavage furrow until late telophase. Sarcomeric myosin and myomesin were only partially disassembled in mitotic myocytes from both 2- and 4-day-old animals. The present study showed that the actin-myosin contractile ring was actually formed during the binucleation process of cardiac myocytes. Molecules involved in the latter stages of cytokinesis may be responsible for incomplete cytokinesis during the binucleation process.

Malformations of the liver: some prenatal and postnatal developmental aspects.

The discovery of a large articulated lobe weighing 165.4 g dependent from the anterior margin of the left liver lobe in a 93-year-old female culminated two decades of observation of anomalous liver conditions in anatomic cadavers. When this data was compared with the information previously recorded from a series of 52 perinatal livers, remarkable discrepancies between the adult and perinatal incidences of various anomalous manifestations were noted. Ectopic accessory lobes were very rare in our adult series as well as in the groups reported in literature reviews. However, they were a relatively common occurrence in the perinatal cases, as were also gallbladder bridges of hepatic tissue, hypertrophic papillary lobes, and marked accessory fissures. In contradistinction, marginal accessory lobes were not noticed in any of the perinatal livers. Because the liver undergoes considerable postnatal reformation, it was concluded that most ectopic lobes, gallbladder bridges, hypertrophic caudate lobe extensions, and accessory fissures disappear during postnatal remodeling, whereas marginal accessory lobes may result from retention of the original perinatal boundaries of the anterior liver margins.

Traumatic abducens nerve paresis in a child.

Trauma is a frequent cause for abducens (sixth) nerve paresis in a child, usually attributed to injury along the nerve's course. An unusual focal lesion of the sixth nerve nucleus is described.

Intimal musculature of the lower anterior spinal artery.

STUDY DESIGN: This was a descriptive microscopic investigation of the smooth musculature in the human lower anterior spinal artery using anatomic cadaver tissues with supporting data derived from angiographic studies of neonatal cadavers. OBJECTIVE: To determine the extent and configuration of the intimal musculature in the lower anterior spinal artery and deduce the probable influence it has on the lower spinal cord blood flow, as well as its effects on the axial anastomotic potentials during aortic cross-clamping. SUMMARY OF BACKGROUND DATA: The high incidence of ischemic spinal cord injury after aortic cross-clamping has led to many studies of autoregulation of the spinal cord blood flow, but none have identified the probable vascular mechanisms. METHODS: Spinal tissue blocks that included the mid-thoracic and thoracolumbar anterior spinal artery, and sections of excised anterior spinal artery were dissected from spinal cords of 16 cadavers, refixed in Bouin's solution, paraffin embedded, and microscopically studied in sections processed in Masson's trichrome stain. A radioangiographic study of barium-perfused spines of neonatal cadavers was used to substantiate histologic observations. RESULTS: The smooth muscle of the tunica media of the lower anterior spinal artery is reinforced by a conspicuous longitudinally disposed layer of intimal muscle. At the junction of the arteria medullaris magna and the anterior spinal artery, this muscle forms intimal cushions that can dramatically alter the luminal diameters of the vessels involved. CONCLUSION: The described muscular characteristics of the lower anterior spinal artery indicate it has a previously unsuspected capacity to control extrinsic and intrinsic aspects of the lower spinal cord blood flow, a factor that should be considered in clinical and experimental procedures involving spinal cord collateral circulation.

HIV associated risk factors: a survey of a troubled adolescent population.

The incidence of Acquired Immunodeficiency Syndrome (AIDS) in young adults and the typical incubation period for AIDS suggest that exposure to this disease often occurs in adolescence, a period of life during which risk taking behavior is particularly common. The population of adolescents with mental or behavioral problems and substance abuse problems at the South Dakota Human Services Center were studied by questionnaire and by human immunodeficiency virus (HIV) screening to assess the current prevalence of seropositivity and the potential for HIV transmission in these troubled youths. Sexual behavior, number of partners, prior incarceration, history of sexual abuse, drug and alcohol use, and knowledge about HIV transmission were examined. While no cases of HIV seropositivity were discovered, the authors are disturbed by the prevalence of known and suspected correlate behaviors and historical traits for HIV infection in this population. Implications for intervention and education are discussed.

Changes in red nucleus neuronal development following maternal alcohol exposure.

The red nucleus of Swiss Webster mouse fetuses was examined for morphological changes following maternal ethanol exposure. Pregnant females were given a liquid diet containing 30% or 0% ethanol-derived calories. Changes in numerical density of neurons and in neuronal nuclear volume were found in the rostral red (RR) nucleus of ethanol-exposed pups but not in the caudal red (CR) nucleus. Because of the integrative nature of the RR, changes in neuronal morphology that might relate to synaptic connections could affect the behavioral response mechanisms of these offspring.

Neuron development in the superior colliculus of the fetal mouse following maternal alcohol exposure.

Pregnant Swiss Webster mice were given a liquid diet with ethanol (EtOH) or isocaloric amounts of maltose dextrin on gestation day (GD) 0 through 18. On GD 18, maternal blood samples were obtained. Fetuses were then removed and fetal brains were prepared for light microscopy. Fetal weight was reduced in the EtOH-exposed group. The ratio of midbrain cross sectional area to cerebral aqueduct was reduced in the ethanol group, while the density of neuronal nuclear population in both the dense outer layer (DS) and sparse inner layer (SS) of the developing superior colliculus was increased. Mean nuclear volume was decreased in the SS.

Persistent left hepatic venous connection with the coronary sinus.

An anomalous direct connection between the left hepatic vein and the coronary sinus in two cadavers is described. The normal development of the central systemic venous system with particular reference to the hepatic region is reviewed and a possible explanation for the present configuration is put forth. Although this configuration of venous anomaly without abnormal shunting of blood is considered clinically insignificant, the association of cardiac arrhythmias with these variations heightens the importance of their identification.

A dose-response curve for human growth hormone.

Although human GH (hGH) has been administered to GH-deficient patients for over 20 yr, there are minimal published data on the relationship of response to dose. We have given hGH on the basis of body weight to 93 prepubertal GH-deficient patients over an initial 12 months of therapy. Their annual growth rate while receiving hGH was 5.58 +/- 2.30 (+/- SD) cm at a dose of 30 mIU/kg, three times a week (tiw; n = 27); 7.31 +/- 1.75 (+/- SD) cm at a dose of 60 mIU/kg, tiw (n = 38); 7.22 +/- 3.12 (+/- SD) cm at a dose of 80 mIU/kg, tiw (n = 12); and 8.94 +/- 1.19 cm (+/- SD) at a dose of 100 mIU/kg, tiw (n = 16). Doubling the dose from 30 to 60 mIU increased the mean rate of growth 1.3 times, and increasing the 30 mIU dose by a factor of 3.3 increased the mean rate of growth 1.6 times. The response (y) as a function of the log-dose (x) is defined by the equation y = -3.12 + 5.80 log x. When the effect of hGH is expressed as the increase in growth rate while receiving therapy, the log-dose relationship is defined by the equation y = -6.09 + 5.67 log x. This dose-response curve provides data which are useful in choosing the best dose of hGH for an individual patient. It also allows a more accurate projection of the costs and benefits of hGH therapy.

Persistent left superior vena cava and associated structural and functional considerations.

Potentially significant associations are presented between anomalous systemic venous return (including both left superior vena cava and left hepatic venous drainage to the coronary sinus), a history of atrial fibrillation and a forme fruste of cor triatriatum in an elderly woman. Lack of associated structural defects or functional deficits makes it difficult to assess the frequency of occurrence in the general population of bilateral superior venae cavae in association with a persistent left hepatic vein draining into the coronary sinus. However, the potential for these systemic venous anomalies needs to be considered when unexplained arrhythmias are encountered. More specifically, the size of the coronary sinus needs to be assessed in patients with arrhythmia.