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The novel rhomboid-like protein RHBDD2 is distantly related to rhomboid proteins, a group of highly specialized membrane-bound proteases that catalyze regulated intramembrane proteolysis. In retina, RHBDD2 is expressed from embryonic stages to adulthood, and its levels show age-dependent changes. RHBDD2 is distinctly abundant in the perinuclear region of cells, and it localizes to their Golgi. A glycine zipper motif present in one of the transmembrane domains of RHBDD2 is important for its packing into the Golgi membranes. Its deletion causes dislodgment of RHBDD2 from the Golgi. A specific antibody against RHBDD2 recognizes two forms of the protein, one with low (39 kDa; RHBDD2(L)) and the other with high (117 kDa; RHBDD2H) molecular masses in mouse retinal extracts. RHBDD2(L) seems to be ubiquitously expressed in all retinal cells. In contrast, RHBDD2H seems to be present only in the outer segments of cone photoreceptors and may correspond to a homotrimer of RHBDD2(L). This protein consistently co-localizes with S- and M-types of cone opsins. We identified a homozygous mutation in the human RHBDD2 gene, R85H, that co-segregates with disease in affected members of a family with autosomal recessive retinitis pigmentosa. Our findings suggest that the RHBDD2 protein plays important roles in the development and normal function of the retina.

It is often recognized, but seldom addressed, that a quantitative assessment of the cumulative effects, both additive and non-additive, of multiple stressors on fish survival would provide a more realistic representation of the factors that influence fish migration. This review presents a compilation of analytical methods applied to a well-studied fish migration, a more general review of quantitative multivariable methods, and a synthesis on how to apply new analytical techniques in fish migration studies. A compilation of adult migration papers from Fraser River sockeye salmon Oncorhynchus nerka revealed a limited number of multivariable methods being applied and the sub-optimal reliance on univariable methods for multivariable problems. The literature review of fisheries science, general biology and medicine identified a large number of alternative methods for dealing with cumulative effects, with a limited number of techniques being used in fish migration studies. An evaluation of the different methods revealed that certain classes of multivariable analyses will probably prove useful in future assessments of cumulative effects on fish migration. This overview and evaluation of quantitative methods gathered from the disparate fields should serve as a primer for anyone seeking to quantify cumulative effects on fish migration survival.

Gestational lead exposure (GLE) produces supernormal scotopic electroretinograms (ERG) in children, monkeys and rats, and a novel retinal phenotype characterized by an increased number of rod photoreceptors and bipolar cells in adult mice and rats. Since the loss of dopaminergic amacrine cells (DA ACs) in GLE monkeys and rats contributes to supernormal ERGs, the retinal DA system was analyzed in mice following GLE. C57BL/6 female mice were exposed to low (27 ppm), moderate (55 ppm) or high (109 ppm) lead throughout gestation and until postnatal day 10 (PN10). Blood [Pb] in control, low-, moderate- and high-dose GLE was ≤ 1, ≤ 10, ~25 and ~40 μg/dL, respectively, on PN10 and by PN30 all were ≤ 1 μg/dL. At PN60, confocal-stereology studies used vertical sections and wholemounts to characterize tyrosine hydroxylase (TH) expression and the number of DA and other ACs. GLE dose-dependently and selectively decreased the number of TH-immunoreactive (IR) DA ACs and their synaptic plexus without affecting GABAergic, glycinergic or cholinergic ACs. Immunoblots and confocal revealed dose-dependent decreases in retinal TH protein expression and content, although monoamine oxidase-A protein and gene expression were unchanged. High-pressure liquid chromatography showed that GLE dose-dependently decreased retinal DA content, its metabolites and DA utilization/release. The mechanism of DA selective vulnerability is unknown. However, a GLE-induced loss/dysfunction of DA ACs during development could increase the number of rods and bipolar cells since DA helps regulate neuronal proliferation, whereas during adulthood it could produce ERG supernormality as well as altered circadian rhythms, dark/light adaptation and spatial contrast sensitivity.

Background Gestational lead exposure (GLE) produces novel and persistent rod-mediated electroretinographic (ERG) supernormality in children and adult animals. Objectives We used our murine GLE model to test the hypothesis that GLE increases the number of neurons in the rod signaling pathway and to determine the cellular mechanisms underlying the phenotype. Results Blood lead concentrations ([BPb]) in controls and after low-, moderate-, and high-dose GLE were ≤ 1, ≤ 10, approximately 25, and approximately 40 μg/dL, respectively, at the end of exposure [postnatal day 10 (PND10)]; by PND30 all [BPb] measures were ≤ 1 μg/dL. Epifluorescent, light, and confocal microscopy studies and Western blots demonstrated that late-born rod photoreceptors and rod and cone bipolar cells (BCs), but not Müller glial cells, increased in a nonmonotonic manner by 16–30% in PND60 GLE offspring. Retinal lamination and the rod:cone BC ratio were not altered. In vivo BrdU (5-bromo-2-deoxyuridine) pulse-labeling and Ki67 labeling of isolated cells from developing mice showed that GLE increased and prolonged retinal progenitor cell proliferation. TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) and confocal studies revealed that GLE did not alter developmental apoptosis or produce retinal injury. BrdU birth-dating and confocal studies confirmed the selective rod and BC increases and showed that the patterns of neurogenesis and gliogenesis were unaltered by GLE. Conclusions Our findings suggest two spatiotemporal components mediated by dysregulation of different extrinsic/intrinsic factors: increased and prolonged cell proliferation and increased neuronal (but not glial) cell fate. These findings have relevance for neurotoxicology, pediatrics, public health, risk assessment, and retinal cell biology because they occurred at clinically relevant [BPb] and correspond with the ERG phenotype.

The adverse effects of low-level developmental lead exposure [blood lead (BPb) ≤ 10 μg/dL] on cognitive, auditory, and visual-motor function are well documented (Canfield et al. 2003; Osman et al. 1999; Rothenberg et al. 2000; Wasserman et al. 2000), but only two studies have examined the impact of low-level developmental Pb exposure on retinal and visual function in children (Altmann et al. 1998; Rothenberg et al. 2002). This lack of research is despite findings that retinal and visual cortical structural–functional abnormalities occur in animals after moderate-level (BPb 11–39 μg/dL) and high-level (BPb ≥ 40 μg/dL) developmental Pb exposure (reviewed by Fox and Boyes 2007; Otto and Fox 1993). Persistent rod photoreceptor-mediated (scotopic) electroretinographic (ERG) and behavioral deficits occur in monkeys, rats, and mice after moderate-level and high-level post-natal Pb exposure (PLE) (Fox and Boyes 2007). The scotopic ERG alterations are characterized by decreases in a-wave and b-wave ERG amplitude (subnormality), sensitivity, and temporal resolution. Similar ERG changes occurred in Pb-exposed workers and isolated retinas exposed to Pb2+ (Otto and Fox 1993). In contrast, adult monkeys with high-level lifetime Pb exposure exhibited increased ERG b-wave amplitudes (supernormality) (Lilienthal et al. 1994). Moreover, our prospective epidemiologic study of children with lifetime low-level and moderate-level Pb exposure revealed that only gestational Pb exposure (GLE) showed a significant dose-dependent relationship with supernormal ERG a-waves and b-waves and increased b-wave sensitivity with no change in implicit times (Rothenberg et al. 2002). In experimental animals, supernormal scotopic ERGs are observed after the loss of retinal dopamine (DA) or zinc. For example, the destruction of dopaminergic amacrine cells by the neurotoxin 6-hydroxydopamine produced supernormal scotopic ERGs with normal implicit times (Olivier et al. 1986; Skrandies and Wassle 1988). Similarly, animals administered nonselective dopamine receptor antagonists had supernormal scotopic b-waves with normal implicit times (Jagadeesh et al. 1980; Schneider and Zrenner 1991). A persistent decrease in retinal tyrosine hydroxylase immunoreactivity after lifetime Pb exposure in monkeys suggests that a loss of DA produced supernormal ERGs (Kohler et al. 1997; Lilienthal et al. 1994). Chelation of retinal Zn produced supernormal scotopic ERGs and increased b-wave sensitivity (Redenti and Chappell 2003). Although no published reports have shown that scotopic ERG supernormality results from an increased number of rods, the log scotopic b-wave threshold and amplitude are linearly related to the number of rod photoreceptors and their rhodopsin content per eye (Dowling 1960). Therefore, we hypothesized that GLE produced supernormal scotopic ERGs by one of these three relatively independent mechanisms: an increased number of rod photoreceptors, decreased retinal DA metabolism, and/or decreased retinal Zn concentrations. To determine the sites and mechanisms of the GLE-induced supernormal scotopic ERGs, we developed a new dose–response model of GLE. Then, we conducted scotopic ERG studies and retinal histologic, morphometric, and neurochemical experiments in adult offspring. Low-level and moderate-level GLE produced supernormal scotopic ERGs, an increased neurogenesis of rods and rod bipolar cells (BCs), and decreased DA synthesis and utilization/release in the absence of retinal injury. GLE produced inverted U-shaped dose–response curves as the high-level GLE dose produced rod cell loss and thus ERG subnormality.

BACKGROUND Transforming growth factor (TGF)-beta2 can produce effective pleurodesis in animals, but its efficacy has not been compared with commonly used pleurodesing agents in sheep, which have a thick pleura resembling that of humans. The acute physiological effects and the level of systemic TGF-beta absorption after its intrapleural administration have not been studied. The aims of the present study were to compare: (i) the effectiveness of TGF-beta2, talc and bleomycin in producing pleurodesis in sheep; (ii) the acute side-effects and systemic TGF-beta levels following the intrapleural administration of these agents; and (iii) histological changes after intrapleural injections of these agents. METHODOLOGY Twelve sheep were divided into three groups and were given a single intrapleural dose of TGF-beta2 (0.25 microg/kg), talc slurry (5 g) or bleomycin (60 IU) via a chest tube. Saline or buffer was injected into the contralateral side, which served as the control. Arterial blood gases and respiratory and heart rates were monitored for the first 24 h. Plasma levels of TGF-beta1 and TGF-beta2 were measured. Pleurodesis was graded macroscopically from 1 (none) to 8 (symphysis > 50% of hemithorax) at day 14. RESULTS At day 14, the pleurodesis score of the TGF-beta2 group (7.7+/-0.6) was similar to that of the talc (7.0+/-1.7) group and significantly higher than that of the bleomycin group (3.3+/-2.3; P < 0.05). No significant differences were seen in arterial blood gas analysis, vital signs and plasma TGF-beta1 and TGF-beta2 concentrations among the three groups. CONCLUSIONS Transforming growth factor-beta2 was as effective as talc and more so than bleomycin in inducing pleurodesis in sheep. Intrapleural administration of TGF-beta2 appeared safe. No acute changes in gaseous exchange or macroscopic abnormalities were seen following intrapleural TGF-beta2. Importantly, there was no evidence of an increase in systemic TGF-beta levels following its intrapleural administration.

Summary: A synthetic peptide (peptide 10) representing a surface-exposed, linear B cell epitope from outer-membrane (OM) protein F of Pseudomonas aeruginosa was shown previously to afford protection in mice from P. aeruginosa infection. This peptide was expressed in tandem with the protein F peptide 18 on each of the two coat proteins of cowpea mosaic virus (CPMV). The chimaeric virus particles (CVPs) expressing the peptides on the S (small) coat protein (CPMV-PAE4) and L (large) coat protein (CPMV-PAE5) were used to immunize mice. Following subcutaneous immunization in Freund's and QuilA adjuvants, CPMV-PAE4 induced antibodies predominantly against peptide 18, whereas CPMV-PAE5 produced antibodies exclusively against peptide 10, indicating that the site of peptide expression on CPMV influences its immune recognition. The anti-peptide antibodies elicited by CPMV-PAE5 were predominantly of the IgG2a isotype, indicating a highly polarized TH1-type response. The peptide-specific IgG2a strongly recognized the whole F protein, but more importantly, recognized protein F in all seven Fisher-Devlin immunotypes of P. aeruginosa. Furthermore, the peptide-specific IgG2a in CVP/QS-21 adjuvant-immunized mice was shown to bind complement and to augment phagocytosis of P. aeruginosa by human neutrophils in vitro. The ability of CPMV-PAE5 to induce P. aeruginosa-specific opsonic IgG2a gives it potential for further development as a protective vaccine against P. aeruginosa.

Previous studies showed that the HIV-1 envelope (Env) protein was not incorporated into vesicular stomatitis virus (VSV) virions unless its cytoplasmic tail was replaced with that of the VSV glycoprotein (G). To determine whether the G tail provided a positive incorporation signal for Env, or if sequences in the Env tail prevented incorporation, we generated mutants of Env with its 150-amino-acid tail shortened to 29, 10, or 3 amino acids (Envtr mutants). Cells infected with VSV recombinants expressing these proteins or an Env-G tail hybrid showed similar amounts of Env protein at the surface. The Env-G tail hybrid or the Envtr3 mutant were incorporated at the highest levels into budding VSV virions. In contrast, the Envtr29 or Envtr10 mutants were incorporated poorly. These results defined a signal preventing incorporation within the 10 membrane-proximal amino acids of the Env tail. Confocal microscopy revealed that this signal functioned by causing localization of human immunodeficiency virus type 1 Env to plasma membrane domains distinct from the VSV budding sites, where VSV proteins were concentrated.

We generated replication-competent, recombinant vesicular stomatitis viruses (VSVs) expressing the human immunodeficiency virus (HIV) envelope protein or an HIV-VSV chimeric envelope protein in which the cytoplasmic domain of the HIV envelope protein was replaced with that from the VSV glycoprotein (G). These recombinants were generated with HIV type 1 (HIV-1) envelopes from both laboratory and primary isolates of HIV-1. The replication-competent recombinant viruses were stable and expressed the foreign proteins at high levels from extra transcription units in VSV. The foreign proteins were processed appropriately and transported to the cell surface. The incorporation of HIV gp120 into VSV particles was demonstrated biochemically only for the construct expressing the chimeric envelopes containing the VSV G cytoplasmic domain. The incorporation of the chimeric HIV envelope protein into the membrane of the recombinant VSV was also demonstrated by electron microscopy with gold-conjugated antibodies. To determine whether specific infection of CD4-positive cells could be demonstrated for these recombinants, we neutralized VSV infectivity due to VSV glycoprotein with anti-VSV serum. The neutralized recombinants expressing the chimeric envelope were able to infect only HeLa cells expressing CD4, and this CD4-specific infectivity was neutralized with anti-HIV serum. This assay also detected a 100-fold-lower titer of CD4-specific infectivity for the VSV recombinant expressing the wild-type HIV envelope. Our results illustrate that it is possible to express functional HIV envelopes from the VSV genome and target the recombinant virus to an alternative receptor. The recombinants may also prove useful as HIV vaccines.

The effect of mercury as Hg2Cl2 and HgCl2 on the antioxidant enzyme levels and its toxicity was investigated in an insect model comprised of adult females of the common housefly, Musca domestica, and fourth-instar larvae of the cabbage looper moth, Trichoplusia ni. HgCl2 was found to be more toxic than Hg2Cl2 to both M. domestica and T. ni. The LC50s for M. domestica were 1.17% and 0.38% w/v concentration for Hg2Cl2 and HgCl2, respectively. For the more tolerant T. ni, the LC50S were 5.15% for Hg2Cl2 and 0.96% w/w concentration for HgCl2. The minimally acute LC5 dose of both oxidation states of Hg was approximately 0.005% for both insects (w/v for M. domestica and w/w for T. ni). At the LC5, both forms of Hg significantly induced the activity of superoxide dismutase in both insect species. Catalase was induced by both Hg2Cl2 and HgCl2 in M. domestica but was only induced by HgCl2 in T. ni. Glutathione-S-transferase, its peroxidase activity, and glutathione reductase activities were also significantly altered in most cases by Hg in both insects although the pattern of alternation was different between the two insects. It is evident that mercury induces oxidative stress in insects as it does in vertebrates. Our findings suggest that insects may serve as a valuable, non-mammalian model species to assess Hg-induced oxidative stress as a component of environmental toxicity.

Removal of lead from gasoline and other environmental sources has decreased the median blood lead concentration ([BPb]) of children in the United States < 10 μg/dL: the current low level of concern [Centers for Disease Control and Prevention (CDC) 1991]. However, there is compelling cross-sectional and prospective epidemiological evidence that [BPb] in children ≤ 10 μg/dL causes cognitive decline (Canfield et al. 2003; Hu et al. 2006; Lanphear et al. 2005; Rothenberg et al. 2002; Winneke et al. 1990). Recently, Gilbert and Weiss (2006) suggested that the BPb action level in children should be 2 μg/dL. Developmental lead exposure also has been linked to a variety of neurological and neurodegenerative disorders in children and adolescents, including attention deficit hyperactivity disorder (ADHD) (Braun et al. 2006), auditory and language impairments (Dietrich et al. 1992; Rothenberg et al. 2000; Yuan et al. 2006), retinal deficits (Rothenberg et al. 2002), neuromotor dysfunction (Bhattacharya et al. 1990, 2006; Ris et al. 2004), and schizophrenia (Opler et al. 2004). Few studies, however, have examined the long-term effects of low-level gestational lead exposure (GLE) despite findings that children with prenatal lead exposure have reduced cognitive functions (Baghurst et al. 1992; Hu et al. 2006; Schnaas et al. 2006; Wasserman et al. 2000), neuromotor and visual motor dysfunction (Ris et al. 2004; Wasserman et al. 2000), and altered auditory and retinal function (Dietrich et al. 1992; Rothenberg et al. 2000, 2002; Wasserman et al. 2000). Maternal lead exposure results from inhalation, diet, and/or eating in lead-contaminated work areas (Correa et al. 2006; Min et al. 1996). Maternal skeletal bone lead from prior exposure mobilizes during pregnancy and lactation (Manton et al. 2003). Lead easily crosses the placental and mammary barriers (Bornschein et al. 1977; Korpela et al. 1986). Thus, the developing fetus and child are at risk, as evidenced by findings that fetal and maternal [BPb] are similar (Korpela et al. 1986). The adverse cognitive consequences of prenatal and postnatal exposure to moderate-level ([BPb] 11–39 μg/dL) and high-level ([BPb] ≥ 40 μg/dL) lead have been studied in rodents (Cory-Slechta 1997; Crofton et al. 1980; Kuhlmann et al. 1997; Wasserman et al. 2000). Several reports also link moderate-to-high-level lead exposure to altered motor activity (Crofton et al. 1980; Ma et al. 1999) and dopaminergic signaling (Antonio and Leret 2000; Cory-Slechta 1997). To date, there are no experimental studies on the effects and mechanisms of low-level GLE on neuromotor function, despite evidence that low-level lead exposure produces these deficits in children (vide supra). In this report we present a new model of human equivalent GLE and the sex-specific physiological, behavioral, and neurochemical abnormalities in year-old GLE mice. These studies were conducted because the long-term consequences of GLE are unknown and increasing evidence indicates that early developmental exposure to neurotoxicants accelerates age-related functional decline and/or produces delayed neurotoxicity (Barone et al. 1995; Basha et al. 2005; Landrigan et al. 2005; Newland and Rasmussen 2000; Rice and Barone 2000; Weiss 1990; Weiss et al. 2002). Sex differences were examined because a) early developmental lead exposure produces a heightened risk for attention, visual motor, and fine-motor deficits in males (Bhattacharya et al. 1990, 2006; Ris et al. 2004); b) male and female animals exhibit differences in exposure and susceptibility to chemicals and lead neurotoxicity (Cory-Slechta et al. 2004; Vahter et al. 2007); and c) this is an important and underexplored area of toxicology (Vahter et al. 2007). Our results show that GLE produced age-related, sex-specific, and non-monotonic dose–response alterations.

Soybean [Glycine max (L.) Merr.] cultivars show differences in their resistance to both the leaf scorch and root rot of sudden death syndrome (SDS). The syndrome is caused by root colonization by Fusarium virguliforme (ex. F. solani f. sp. glycines). Root susceptibility combined with reduced leaf scorch resistance has been associated with resistance to Heterodera glycines HG Type 1.3.6.7 (race 14) of the soybean cyst nematode (SCN). In contrast, the rhg1 locus underlying resistance to Hg Type 0 was found clustered with three loci for resistance to SDS leaf scorch and one for root infection. The aims of this study were to compare the inheritance of resistance to leaf scorch and root infection in a population that segregated for resistance to SCN and to identify the underlying quantitative trait loci (QTL). “Hartwig”, a cultivar partially resistant to SDS leaf scorch, F. virguliforme root infection and SCN HG Type 1.3.6.7 was crossed with the partially susceptible cultivar “Flyer”. Ninety-two F5-derived recombinant inbred lines and 144 markers were used for map development. Four QTL found in earlier studies were confirmed. One contributed resistance to leaf scorch on linkage group (LG) C2 (Satt277; P = 0.004, R 2 = 15%). Two on LG G underlay root infection at R8 (Satt038; P = 0.0001 R 2 = 28.1%; Satt115; P = 0.003, R 2 = 12.9%). The marker Satt038 was linked to rhg1 underlying resistance to SCN Hg Type 0. The fourth QTL was on LG D2 underlying resistance to root infection at R6 (Satt574; P = 0.001, R 2 = 10%). That QTL was in an interval previously associated with resistance to both SDS leaf scorch and SCN Hg Type 1.3.6.7. The QTL showed repulsion linkage with resistance to SCN that may explain the relative susceptibility to SDS of some SCN resistant cultivars. One additional QTL was discovered on LG G underlying resistance to SDS leaf scorch measured by disease index (Satt130; P = 0.003, R 2 = 13%). The loci and markers will provide tagged alleles with which to improve the breeding of cultivars combining resistances to SDS leaf scorch, root infection and SCN HG Type 1.3.6.7.

Previously, we investigated ubisemiquinone (SQ) EPR spectra associated with NADH-ubiquinone oxidoreductase (complex I) in the tightly coupled bovine heart submitochondrial particles (SMP). Based upon their widely differing spin relaxation rate, we distinguished SQ spectra arising from three distinct SQ species, namely SQNf (fast), SQNs (slow), and SQNx (very slow). The SQNf signal was observed only in the presence of the proton electrochemical gradient ( Δ μ H + ) , while SQNs and SQNx species did not require the presence of Δ μ H + . We have now succeeded in characterizing the redox and EPR properties of SQ species in the isolated bovine heart complex I. The potentiometric redox titration of the gz,y,x = 2.00 semiquinone signal gave the redox midpoint potential (Em) at pH 7.8 for the first electron transfer step [Em1(Q/SQ)] of −45 mV and the second step [Em2(SQ/QH2)] of −63 mV. It can also be expressed as [Em(Q/QH2)] of −54 mV for the overall two electron transfer with a stability constant (Kstab) of the SQ form as 2.0. These characteristics revealed the existence of a thermodynamically stable intermediate redox state, which allows this protein-associated quinone to function as a converter between n = 1 and n = 2 electron transfer steps. The EPR spectrum of the SQ species in complex I exhibits a Gaussian-type spectrum with the peak-to-peak line width of ∼6.1 G at the sample temperature of 173 K. This indicates that the SQ species is in an anionic Q − state in the physiological pH range. The spin relaxation rate of the SQ species in isolated complex I is much slower than the SQ counterparts in the complex I in situ in SMP. We tentatively assigned slow relaxing anionic SQ species as SQNs, based on the monophasic power saturation profile and several fold increase of its spin relaxation rate in the presence of reduced cluster N2. The current study also suggests that the very slowly relaxing SQNx species may not be an intrinsic complex I component. The functional role of SQNs is further discussed in connection with the SQNf species defined in SMP in situ.

BACKGROUND We have recently demonstrated that transforming growth factor beta-2 (TGF-beta2) can produce effective pleurodesis. Whether this effect can be reproduced by the use of its downstream proteins is not known. This study compared the effectiveness of TGF-beta2 and fibronectin in inducing pleurodesis in rabbits. METHODOLOGY New Zealand white rabbits (1.5-2.0 kg) were given 1.7 microg of TGF-beta2 (n=5) or 2.0 mg of cellular fibronectin (n=4) intrapleurally via a chest tube. The induced pleural fluid was collected and analyzed. The rabbits were sacrificed after 14 days. The pleurodesis was graded macroscopically from 1 (none) to 8 (symphysis > 50%). RESULTS All rabbits in the TGF-beta2 group developed effective pleurodesis while none in the fibronectin group had scores > 2 (pleurodesis scores 7.0 +/- 0.6 vs 1.3 +/- 0.3, P < 0.001). Rabbits that received TGF-beta2 produced large amounts of pleural fluid initially (< 4 days). Microscopically, the pleura of rabbits in the TGF-beta2 group showed prominent spindle cell proliferation and collagen deposition, but no significant inflammation or mesothelial proliferation. Pleural tissues of rabbits in the fibronectin group had occasional thin collagen deposits only. The intrapleural administration of 2.0 mg of fibronectin, a downstream product of TGF-beta, did not induce effective pleurodesis, as did the intrapleural administration of TGF-beta2. CONCLUSIONS The present study suggests that the mechanism by which TGF-beta2 induces pleurodesis is not predominantly dependent on the production of fibronectin.

The 3.0 A resolution crystal structure of Pariacoto virus (PaV) reveals extensive interactions between portions of the viral RNA genome and the icosahedral capsid. Under the protein shell of the T = 3 quasi equivalent capsid lies a dodecahedral cage composed of RNA duplex that accounts for approximately 35% of the single-stranded RNA genome. The highly basic N-terminal regions (residues 7-54) of the subunits, forming pentamers (A subunits) are clearly visible in the density map and make numerous interactions with the RNA cage. The C-terminal segments (residues 394-401) of the A subunits lie in channels near the quasi three-fold axes. Electron cryo-microscopy and image reconstruction of PaV particles clearly show the dodecahedral RNA cage.

An assembly intermediate of a small, non-enveloped RNA virus has been discovered that exhibits striking differences from the mature virion. Virus-like particles (VLPs) of Nudaurelia capensis omega virus (NomegaV), a T=4 icosahedral virus infecting Lepidoptera insects, were produced in insect cells using a baculovirus vector expressing the coat protein. A procapsid form was discovered when NomegaV VLPs were purified at neutral pH conditions. These VLPs were fragile and did not undergo the autoproteolytic maturation that occurs in the infectious virus. Electron cryo-microscopy (cryoEM) and image analysis showed that, compared with the native virion, the VLPs were 16% larger in diameter, more rounded, porous, and contained an additional internal domain. Upon lowering the pH to 5.0, the VLP capsids became structurally indistinguishable from the authentic virion and the subunits autoproteolyzed. The NomegaV protein subunit coordinates, which were previously determined crystallographically, were modelled into the 28 A resolution cryoEM map of the procapsid. The resulting pseudo-atomic model of the NomegaV procapsid demonstrated the large rearrangements in quaternary and tertiary structure needed for the maturation of the VLPs and presumably of the virus. Based on this model, we propose that electrostatically driven rearrangements of interior helical regions are responsible for the large conformational change. These results are surprising because large structural rearrangements have not been found in the maturation of any other small RNA viruses. However, similarities of this conformational change to the maturational processes of more complex DNA viruses (e.g. bacteriophages and herpesvirus) and to the swelling of simple plant viruses suggest that structural changes in icosahedral viruses, which are integral to their function, have similar strategies and perhaps mechanisms.

The influence of peptide structure on immunogenicity has been investigated by constructing a series of cowpea mosaic virus (CPMV) chimaeras expressing the 14 amino acid NIm-1A epitope from human rhinovirus 14 (HRV-14) at different positions on the capsid surface. Biochemical and crystallographic analysis of a CPMV/HRV chimaera expressing the NIm-1A epitope inserted into the betaC'-betaC" loop of the S protein revealed that, although the inserted peptide was free at its C-terminus, it adopted a conformation distinct from that previously found when a similarly cleaved peptide was expressed in the betaB-betaC loop of the S protein. Adjustment of the site of insertion within the betaB-betaC loop resulted in the isolation of a chimaera in which cleavage at the C-terminus of the epitope was much reduced. Crystallographic analysis confirmed that in this case the epitope was presented as a closed loop. Polyclonal antisera raised against the CPMV/ HRV chimaera presenting the NIm-1A epitope as a closed loop had a significantly enhanced ability to bind to intact HRV-14 particles compared with antisera raised against chimaeras presenting the same sequence as peptides with free C-termini. These results demonstrate that the mode of presentation of an epitope on a heterologous carrier can dramatically affect its immunological properties.

T cell function is a critical determinant of immune responses as well as susceptibility to allergic diseases. Activated T cells can differentiate into effectors whose cytokine profile is limited to type 1 (IFN-gamma-dominant) or type 2 (IL-4-, IL-5-dominant) patterns. To investigate mechanisms that connect extracellular stimuli with the regulation of effector T cell function, we have measured immune responses of transgenic mice whose NF-kappa B/Rel signaling pathway is inhibited in T cells. Surprisingly, these mice developed type 2 T cell-dependent responses (IgE and eosinophil recruitment) in a model of allergic pulmonary inflammation. In contrast, type 1 T cell responses were severely impaired, as evidenced by markedly diminished delayed-type hypersensitivity responses, IFN-gamma production, and Ag-specific IgG2a levels. Taken together, these data indicate that inhibition of NF-kappa B can lead to preferential impairment of type 1 as compared with type 2 T cell-dependent responses.

We describe a replication-competent, recombinant vesicular stomatitis virus (VSV) in which the gene encoding the single transmembrane glycoprotein (G) was deleted and replaced by an env -G hybrid gene encoding the extracellular and transmembrane domains of a human immunodeficiency virus type 1 (HIV-1) envelope protein fused to the cytoplasmic domain of VSV G. An additional gene encoding a green fluorescent protein was added to permit rapid detection of infection. This novel surrogate virus infected and propagated on cells expressing the HIV receptor CD4 and coreceptor CXCR4. Infection was blocked by SDF-1, the ligand for CXCR4, by antibody to CD4 and by HIV-neutralizing antibody. This virus, unlike VSV, entered cells by a pH-independent pathway and thus supports a pH-independent pathway of HIV entry. Additional recombinants carrying hybrid env -G genes derived from R5 or X4R5 HIV strains also showed the coreceptor specificities of the HIV strains from which they were derived. These surrogate viruses provide a simple and rapid assay for HIV-neutralizing antibodies as well as a rapid screen for molecules that would interfere with any stage of HIV binding or entry. The viruses might also be useful as HIV vaccines. Our results suggest wide applications of other surrogate viruses based on VSV.

Lucis is a software program that allows the manipulation of images through the process of selective contrast pattern emphasis. Using an image-processing algorithm called Differential Hysteresis Processing (DHP), Lucis extracts and highlights patterns based on variations in image intensity (luminance). The result is that details can be seen that would otherwise be hidden in deep shadow or excessive brightness. The software is contained on a single floppy disk, is easy to install on a PC, simple to use, and runs on Windows 95, Windows 98, and Windows NT operating systems. The cost is $8,500 for a license, but is estimated to save a great deal of money in photographic materials, time, and labor that would have otherwise been spent in the darkroom. Superb images are easily obtained from unstained (no lead or uranium) sections, and stored image files sent to laser printers are of publication quality. The software can be used not only for all types of microscopy, including color fluorescence light microscopy, biological and materials science electron microscopy (TEM and SEM), but will be beneficial in medicine, such as X-ray films (pending approval by the FDA), and in the arts.

It has been proposed that the domain of the regulatory enzyme, CTP:phosphocholine cytidylyltransferase, which mediates reversible binding of the enzyme to membranes, is an amphipathic alpha-helix of approximately 60 amino acid residues and that this domain is adjacent to the putative active site domain of this enzyme. Circular dichroism indicated that the secondary structures of two overlapping peptides spanning this region were predominantly alpha-helical in the presence of PG vesicles or sodium dodecyl sulfate micelles. Interproton distances were obtained from two-dimensional NMR spectroscopic measurements to solve the structures of these two peptides. The C-terminal 22 amino acid peptide segment (corresponding to Val267-Ser288) was a well-defined alpha-helix over its length. The N-terminal 33-mer (corresponding to Asn236-Glu268) was composed of an alpha-helix from Glu243 to Lys266, a well-structured bend of about 50 degrees at Tyr240-His241-Leu242, and an N-terminal four-residue helix. It is proposed that the three residues involved in generating the bend act as the hinge between the catalytic and regulatory domains. The nonpolar faces of the 33-mer and 22-mer were interrupted by Ser260, Ser271, and Ser282. These residues may serve to limit the hydrophobicity and facilitate reversible and lipid-selective membrane binding. The hydrophobic faces of the helices were flanked by a set of basic amino acid residues on one side and basic amino acid residues interspersed with glutamates on the other. The disposition of these side chains gives clues to the basis for the specificities of these peptides for anionic surfaces.

The expression of novel TrkB receptor transcripts has been characterized to understand the potentially diverse roles of brain- derived neurotrophic factor (BDNF) in the developing avian visual system. In situ localization with an extracellular domain probe common to all TrkB transcripts labeled a sub-population of large retinal ganglion cells as well as many associated visual nuclei, including the neuronal layers within the tectum that receive retinal innervation. Because of the potential for structurally and functionally distinct receptors derived from the TrkB gene locus, cDNA cloning and reverse transcription-PCR analysis were used to further analyze receptor isoform expression in the retina and tectum. Receptor isoforms were sequenced that contained a deletion of the N terminus, a deletion in the putative ligand-binding domain, or a deletion in the cytoplasmic juxtamembrane (JM) domain. Two novel JM insertion sequences also were identified, one of which exhibits weak homology to beta-actin and was found in both kinase-containing (TK+) and kinase deletion (KD) receptor isoforms. In the developing retina, TK+ receptor mRNA is upregulated during the period of retinal ganglion cell (RGC) death, consistent with the proposed role of BDNF as a tectal-derived survival factor for RGCs. However, the expression of TK+ transcripts in the tectum indicates that this structure also contains cells responsive to BDNF throughout development. Because BDNF is expressed in both the retina and tectum, it is conceivable that TrkB also mediates autocrine/paracrine signaling within these structures or anterograde retinotectal trophic support.

Animal physiology graduate students provide an excellent personnel resource for laboratories performing human assisted reproductive technology (ART) procedures. However, the basic training of these students falls short of what is required for this highly specialized field. We designed a course to enhance their education in this area via classroom and hands-on laboratory instruction in a hospital and university setting. Topics covered in the course included in vitro maturation, in vitro fertilization, embryo culture, embryo transfer, quality control, quality assurance, micromanipulation, and cryopreservation. These techniques were applied to a group project to evaluate the influence of spermatozoal quality and quantity on early embryonic development in cattle and humans. Student grades were based on 1) oral and written examinations; 2) demonstrated competency in laboratory techniques; 3) presentation of class project data at a state academy of science meeting; and 4) initiative, determination, and interest in the coursework. Three aspects of the course stood out as very positive. First, the team approach to accomplishing a class project was new to some of the graduate students. Second, a bond was formed between hospital- and university-based faculty that did and will continue to foster unique teaching and research opportunities between the two groups. Third, the opportunity for students to present research data in a formal setting was very rewarding. This course made the students keenly aware of the many aspects of ART and provided them with specialized skills that should make them more marketable in the field of reproductive technology.

The potential usefulness of an insect model to evaluate oxidative stress induced by environmental pollutants was examined with trivalent arsenic (As3+, NaAsO2) and pentavalent arsenic (As5+, Na2HAsO4) in adult female house flies, Musca domestica, and fourth-instar cabbage loopers, Trichoplusia ni. M. domestica was highly susceptible to both forms of arsenic following 48 h exposure in the drinking water with LC50s of 0.008 and 0.011% w/v for As3+ and As5+, respectively. T. ni larvae were susceptible to dietary As3+ with an LC50 of 0.032% w/w but seem to tolerate As5+ well with an LC50 of 0.794% concentration after 48 h exposure. The minimally acute LC5 dose of both As3+ and As5+ varied considerably but averaged 0.005% for both insects. The potential of both valencies of arsenic for inducing oxidative stress in the insects exposed ad libitum to approximately LC5 levels was assessed. The parameters examined were the alterations of the antioxidant enzyme activities of superoxide dismutase (SOD), catalase (CAT), glutathione transferase (GST), the peroxidase activity of glutathione transferase (GSTPX), and glutathione reductase (GR), and increases in lipid peroxidation and protein oxidation. SOD (1.3-fold), GST (1.6-fold), and GR (1.5-fold) were induced by As3+ in M. domestica but CAT and GSTPX were not affected. As5+ had no effect on M. domestica. In T. ni, the antioxidant enzyme activities were not affected by As3+ except for SOD which was suppressed by 29.4% and GST which was induced by 1.4-fold. As5+ had no effect except the suppression of SOD by 41.2%. Lipid peroxidation and protein oxidation, which represent stronger indices of oxidative stress, were elevated in both insects by up to 2.9-fold. However, based on the antioxidant enzyme response to the arsenic anions, the mode of action of arsenic induced oxidative stress may differ between the two insects. Until this aspect is further clarified, evidence at this time favors the prospect of As3+ as a pro-oxidant, especially for M. domestica.

Limited chymotrypsin proteolysis of CTP:phosphocholine cytidylyltransferase (CT; EC 2.7.7.15) produced several distinct fragments which were mapped to the N terminus of CT using antibodies directed against the N and C terminus and the conserved central domain. A time course of chymotrypsin proteolysis showed a progression in digestion as follows: 42--39--35--30--28--26 kDa. The binding of CT and of the chymotrypsin fragments to lipid vesicles was assessed by floatation analysis. The ability of the fragments to bind to activating lipid vesicles correlated with the presence of a putative amphipathic alpha-helix, helix-1, between residues 236 and 293. Fragments lacking this helix could, however, bind to phosphatidylcholine/sphingosine vesicles, which inhibit CT activity, and were capable of dimer formation. The degree of resistance to chymotrypsin degradation increased when CT was bound to the strongly activating lipid vesicles phosphatidylcholine/oleic acid (1:1) and phosphatidylcholine/phosphatidylglycerol (1:1). Conversion of the 39- and 35-kDa fragments, which contain the intact helix-1, to the 30-, 28-, and 26-kDa bands, which lack helix-1, required longer proteolysis times, suggesting that this helical domain is more shielded from solvent upon membrane binding. These results support the theory that CT has a bipartite tertiary structure composed of a globular N-terminal domain and an extended C-terminal domain and that CT interacts with membranes via its putative amphipathic helix which intercalates into the membrane bilayer of activating phospholipids.

Flock house virus (FHV) is a small icosahedral insect virus of the family Nodaviridae. Its genome consists of two messenger-sense RNA molecules, both of which are encapsidated in the same particle. RNA1 (3.1 kb) encodes proteins required for viral RNA replication; RNA2 (1.4 kb) encodes protein alpha (43 kDa), the precursor of the coat protein. When Spodoptera frugiperda cells were infected with a recombinant baculovirus containing a cDNA copy of RNA2, coat protein alpha assembled into viruslike precursor particles (provirions) that matured normally by autocatalytic cleavage of protein alpha into polypeptide chains beta (38 kDa) and gamma (5 kDa). The particles were morphologically indistinguishable from authentic FHV and contained RNA derived from the coat protein message. These results showed that RNA1 was required neither for virion assembly nor for maturation of provirions. Expression of mutants in which Asn-363 at the beta-gamma cleavage site of protein alpha was replaced by either aspartate, threonine, or alanine resulted in assembly of particles that were cleavage defective. For two of the mutants, unusual structural features were observed after preparation for electron microscopy. Particles containing Asp at position 363 were labile and showed a strong tendency to break into half-shells. Particles in which Asn-363 was replaced by Ala displayed a distinct hole in an otherwise complete shell. The third mutant, containing Thr at position 363, was indistinguishable in morphology from authentic FHV.

High resolution transmission electron microscopy (TEM) has shown that bovine tau are 2.1 +/- 0.2-nm diameter filaments which are triple-stranded left-hand helical structures composed of three 1.0 +/- 0.2-nm strands. The reported amino acid sequence of human and bovine tau have been computer processed to predict secondary structure. Within the constraints imposed by the images, the secondary structure models and other structural information have been used to calculate tau's maximum and minimum length. The length calculations and secondary structure form the basis for image interpretation. This work indicates that each approximately 1.0-nm strand is a tau polypeptide chain and that the approximately 2.1-nm filament is composed of three separate tau chains (tau3). Bovine tau length measurements indicate that tau trimer filaments are generally longer than a fully extended tau monomer. These measurements indicate that each trimer, tau3, is joined with other trimers to form long tau polymers, (tau3)n. An inverse temperature transition has been found in the circular dichroism spectrum of tau indicating that its structure is less ordered below 20 degrees C and more ordered at 37 degrees C. The implications of this phenomenon with respect to tau's temperature-dependent ability to reconstitute microtubules is discussed and a mechanism for the possible abnormal aggregation of tau into neurofibrillary tangles in Alzheimer's disease is proposed.

Transfer coefficients for 137Cs from the Chernobyl accident were determined for milk (Fm) and meat (Ff) of cows and sheep in Hungary. Fm and Ff for both cows and sheep fed forage harvested within 1 mo of the accident were lower than results reported for worldwide fallout from weapons tests. Forage harvested 60 d or later after the accident produced an Fm similar to results from feeding soluble 134Cs. The results are interpreted to indicate three distinct categories of Fm about 2.0 X 10(-3), 4.0 X 10(-3) and 1.4 X 10(-2) d L-1, respectively, for Chernobyl fallout, worldwide fallout and soluble Cs isotopes or 137Cs contained in plants from soil uptake.

We have hybridized pulse-labeled nuclear transcripts to cloned DNA fragments from the rabbit beta-like globin genes to determine the developmental timing, extent, and asymmetry of their transcription. The fetal-adult gene beta 1 was transcribed in fetal liver but not embryonic nuclei, whereas genes beta 3 and beta 4, which encode embryonic globin polypeptides, were transcribed only in embryonic nuclei. This shows that the switch from embryonic to fetal-adult globin production in rabbits is accomplished primarily by differential transcription of the beta-like globin genes. Gene beta 1 was subdivided into M13 subclones and tested for hybridization to nascent RNA. The nucleotide sequence of the 3' flanking region of gene beta 1 was also determined for 2,447 base pairs past the polyadenylation [poly(A)] site. No transcripts were found 5' to the cap site, but asymmetric transcription of gene beta 1 proceeded at a high level through the gene and past the poly(A) addition site for 603 nucleotides. The level of transcription declined after this, gradually dropping through the next 568 nucleotides. No polymerases were found on a fragment that begins 1,707 nucleotides past the poly(A) site; this fragment was part of a segment of repetitive DNA. These data show that the transcription unit of gene beta 1 begins at or near the cap nucleotide and extends at least 1,171 but no more than 1,706 nucleotides past the poly(A) addition site. The DNA segment that precedes the region of declining transcription contained an inverted repeat and encoded a short RNA transcribed by RNA polymerase II from the strand opposite the beta 1 transcript. These two features may function to attenuate the transcription of gene beta 1. An inverted repeat and a potential polymerase II transcription unit were also found in the homologous segment 3' to the human beta-globin gene. A short DNA segment close to the 3' end of the beta 1 transcription unit was transcribed more actively than the surrounding DNA, and it contained sequences that match the consensus internal control region for RNA polymerase III. This DNA segment may contain a separate polymerase III transcription unit. A member of the D repeat family located 3' to gene beta 1 was not transcribed in its entirety coordinately with beta 1.

The well-documented physiological role of nerve growth factor (NGF) in peripheral sympathetic and neural-crest-derived sensory neurons in vivo has its exact counterpart in vitro. This provided the conceptual basis for developing in vitro analytical procedures for the purification of new neurotrophic molecules. The experimental approaches used are discussed in the context of the purification of new neurotrophic factors, brain-derived neurotrophic factor (BDNF) and ciliary neurotrophic factor (CNTF). The importance of the modulatory role played by extracellular matrix molecules, in particular laminin, on both NGF-mediated and BDNF-mediated survival effects is also delineated. BDNF is a very basic (pI approximately 10) molecule of about 12 kDa, having physico-chemical characteristics close to those of the monomer of NGF. However, the spectrum of its biological actions is distinctly different from that of NGF. In particular, BDNF supports the survival of retinal ganglion cells and placode-derived peripheral sensory neurons which are not supported by NGF. The trophic supply of primary sensory neurons projecting to both the central nervous system and the periphery is discussed. It is hypothesized that sensory neurons receive limited quantities of neurotrophic molecules from both peripheral and central axons, a mechanism ensuring the survival of neurons adequately connected with both peripheral and central targets.

The thoracic flight muscle from adult male NAIDM house flies was examined, from emergence to very old age. Thin sections stained with uranyl acetate and bismuth showed no myofibrillar or mitochondrial degeneration from 1 day to 19 days post-emergence, contrary to earlier reports. Some progressive loss in glycogen content and increase in mitochondrial size were observed for muscle from young to very old flies. However, there was no conclusive evidence of fusion of smaller mitochondria into larger ones with advancing age, despite exhaustive examination of representative sections of muscle samples of adult males of different ages.

Upon exposure to specific antigen in tissue culture, sensitive lymphocytes released macrophage migration inhibition factor and other lymphokines into the supernatant culture medium. Migration of peritoneal macrophages from nonsensitive animals was inhibited in the presence of such supernatants. However, with previous techniques it was found that an inhibitory effect was present at only low low titers (less than 10(2)). It is therfore of great interest that by increasing cellular density, the total number of cells being kept constant, inhibitory activity can be amplified by a factor as great as 10(10). This amplification was observed only when lymphocytes and macrophages were loosely packed, as by spontaneous sedimentation in a conical test tube. The effect was abolished by dispersing the cell suspension in a flat-bottomed flask or, alternatively, by shaking the test tube so that intimate prolonged intercellular contact was prevented.

When tuberculin-sensitive peritoneal exudate cells are incubated in a culture flask with tuberculin purified protein derivative, macrophage inhibition factor and other lymphokines are released into the culture medium. We have described how, if incubation is carried out in a stationary conical culture tube, intercellular contact between the peritoneal exudate cells is facilitated as the cells sediment into a pellicle at the bottom of the tube. This results in augmented release of inhibitory lymphokines into the supernatant culture medium with titers up to 10 9 times greater than those obtained by conventional culture methods using a flatbottomed culture dish or flask. When such high-titered inhibitory supernatants were subjected to fractionation by sequential Amicon ultrafiltration, two clearly distinct macrophage-inhibitory lymphokines were found. The first was present, after fractionation, in a titer of 10 12 , had a molecular weight in the range of 50,000 to 100,000, and was heat stable at 56°C for 1 h. This moiety is probably identical to guinea pig macrophage inhibition factor. Unexpectedly, a second heat-labile inhibitory substance with a molecular weight between 500 and 1,000 was found in a titer of 10 4 after fractionation. This low-molecular-weight, heat-labile material may represent a new lymphokine with a direct inhibitory action on macrophage migration. Theoretically, the data are also consistent with the possibility that it could act as a chemical immunotransmitter which stimulates amplified production of macrophage inhibition factor by lymphocytes within the cell pellicle and leads indirectly to inhibition of macrophage migration.

High potency preparations of a new heat-labile, low m.w. (less than 5000) lymphokine (LMWL) were obtained by culturing tuberculin-sensitive guinea pig peritoneal exudate cells (PEC) with PPD in geometric conditions that promote amplified lymphokine production. This LMWL has the ability, in the presence of PPD, to stimulate nonsensitive PEC to produce a heat-stable molecule(s) resembling MIF with a m.w. in the range 50,000 to 100,000. The effects of the LMWL (less than 5000 daltons) and the MIF-like molecule(s) (50,000 to 100,000 daltons) were defined by the indirect macrophage migration assay and a macrophage deoxyglucose uptake assay. It is possible that LMWL represents a form of transfer factor with the ability to recruit unsensitized lymphocytes to produce MIF.

A study was undertaken to examine, by scanning electron microscopy (SEM), the kidney glomeruli of control mice 1 mo, 10 mo, and 24 mo of age, as well as dietarily restricted mice 10 mo and 24 mo of age. One month old female C57BL/6J mice were offered one of the following: 1) a control diet containing 24% protein fed ad lib; 2) the control diet fed on alternate days (intermittently fed); or 3) a diet containing 4% protein fed ad lib. Animals were sacrificed, by aldehyde perfusion at 1 mo, 10 mo, and 24 mo of age. The kidneys were sliced and prepared for SEM. There was a significant age-related increase in glomerular diameter and amount of microvilli on the podocyte surface (microvillus index). Although the diameters of the podocytes increased approximately 20% with age, these differences were not statistically significant. Feeding a 4% protein diet resulted in smaller diameters of glomeruli and podocytes as well as smaller microvilli indices as compared to those of control animals. Although similar differences were observed in the kidneys of intermittently fed animals, only the microvillus index was statistically significant. Therefore, dietary manipulations, which have been shown to increase life span, result in marked morphological differences when compared to control animals.

Pineal N-acetyltransferase (NAT) activity and radioimmunoassayable levels of melatonin were compared in 2-month-old (young), 12-month-old (middle-aged), and 29-month-old (old) female rats killed at 1600 h (during the light) and at 2300 h (4 h after darkness onset) and 0100 h (6 h after darkness onset). During the light period, NAT levels were equivalent in pineals from each age group. With the onset of darkness NAT levels rose sharply and were again equivalent in all groups at 2300 h. At 0100 h pineal NAT values in the old rats were lower than in the other two groups. Melatonin values were low in pineal glands of all animals killed at 1600 h in light. By 4 h after darkness onset pineal melatonin content in the young rats had increased 12-fold compared to a 6-fold rise in the old animals. Melatonin levels in the middle aged rats were intermediate between the other two groups. Similar relationships were observed in the rats killed at 0100 h. By this time the young rats had melatonin levels 17 times higher than during the day while the increase in the old rats was only 7-fold; 12-month-old rats again had intermediate levels. The finding show a marked reduction in pineal melatonin with aging in female rats.

We have compared the ability of rat liver plasma membranes and isolated hepatocytes to bind and degrade insulin. Isolated cells were prepared in two different ways: by mechanical separation of cells and by coliagenase digestion of extracellular matrix. In all studies the binding and degradative characteristics of both types of hepatocyte preparations were identical. Furthermore, with one exception, the binding characteristics of membranes and cells were also quite similar. The only exception concerned the amount of insulin bound by hepatocytes as compared to liver membranes. Thus, at concentrations of cells (1.2 × 106 cells per milliliter) and membranes (150 µg. protein per milliliter) that gave equal binding at insulin concentrations 100 ng./ml. was greater with use of hepatocytes. Additional studies indicated that, in contrast to membranes, at the higher insulin concentration only 75 percent of the previously bound insulin could be recovered from hepatocytes. Thus, a nondissociable component exists, which probably represents intracellular radioactivity and appears to account for the higher specific insulin binding by cells at higher insulin concentrations. When insulin degradation was studied at the above hepatocyte and plasma membrane concentrations, cells degraded 30 per cent more insulin than did membranes. Kinetic analysis of these data revealed that the Km for insulin degradation (5 × 10−7 M at 37°) was the same for both systems whereas the Vmax was greater with use of hepatocytes. In conclusion: (1) Preparation of hepatocytes by coliagenase digestion does not appear to alter insulin binding or degradation; (2) studies of liver membranes and isolated hepatocytes obtained from normal rats should yield similar information about insulin-receptor interaction as long as insulin concentrations

A 6.5-year-old horse with a history of exercise-induced pulmonary hemorrhage was admitted because of acute onset of epistaxis, dyspnea, high respiratory rate, pale mucous membranes, and dark feces. There was no clinical or laboratory evidence of a bleeding disorder, and the horse's anemia was considered to be secondary to pulmonary hemorrhage. The cause of the hemorrhage was not found on thoracic sonograms or from cytologic examination of transtracheal aspirates. Despite supportive care, the horse's health deteriorated, and it was euthanatized. Necropsy revealed blood in the thoracic and peritoneal cavities and reddish black masses in many tissues. Histologic examination confirmed a diagnosis of hemangiosarcoma. Hemangiosarcoma is a rare tumor in horses; however, as this case demonstrates, it can cause pulmonary hemorrhage and respiratory distress and may be difficult to diagnose before death.

Soft tissue lesions are often secondary to equine carpal injury. The clinical and pathological nature of soft tissue changes following carpal trauma are varied. This report describes a case of intersynovial fistula between the radial carpal joint and the common digital extensor tendon sheath. The differential diagnosis and pathological features of equine soft tissue carpal injuries are discussed.

The defense of the lung against infections, toxins and allergens is accomplished by an excretory transport mechanism and by the interaction of cellular and humoral defense systems. Pulmonary alveolar macrophages represent a common effector pathway for both nonspecific cellular phagocytic defenses and for specifically triggered cell-mediated immunity, via T lymphocytes. Nonspecific activation of macrophages is induced by toxic substances. Studies of the immunocytologic system indicate partial compartmentalization and "local" immunity for both cellular and humoral systems. Further studies on pulmonary cell-mediated immunity have characterized an amplification mechanism by which antigen-induced stimulation of T lymphocytes leads to recruitment of nonsensitive lymphocytes through the production of a low-molecular weight "transfer factor." Other lymphocyte-produced mediators (lymphokines) act to attract, aggregate and accumulate macrophages in areas of inflammation. In addition, macrophages are "activated" and show enhanced microbicidal capabilities as well as enhanced resistance to the cytotoxic effects of certain ingested microorganisms. It is postulated that cellular (nonspecific) and cell-mediated (specific) immune defenses play important roles in protection against several categories of microorganisms in a hierarchy of virulence.

Concentrations of 129I and values of the 129I/127I ratio have been measured in over forty individual thyroids of human, cow and deer from Missouri. Deer thyroids show an average value of 129I/127I = 1.8 × 10−8 and 129I concentrations of 3 × 10−3 pCi per g of thyroid (wet weight). Thyroids of

Antiserum was prepared in rabbits against the A antigen and a new antigen (labeled the D antigen) of yeast-phase cells of Blastomyces dermatitidis by using immunoelectrophoretic precipitin arcs as vaccine. The antiserum can be used as a reference serum in the immunodiffusion test for identyfying antibodies to the A antigen in sera from patients thought to have blastomycosis. Antibodies to the D antigen have not yet been found in human sera. The rabbit antiserum that was absorbed with Histoplasma capsulatum yeast-phase cells did not react in the complement fixation test with either H. capsulatum yeast-phase or mycelial-phase complement fixation antigen. Therefore, it could also be used as a positive control serum in the complement fixation test. A fluorescent-antibody conjugate prepared from the absorbed serum was found to be valuable for the identification of yeast-phase cells of B. dermatitidis.

This collaborative work was undertaken to resolve discrepancies in reports of the number of forms of complement component C6 present in the circulation of mice from various inbred strains. Plasma C6 was analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and by isoelectric focusing (IEF), and C6 band patterns were developed by electroblotting and immunoprobing. Results of C6 allotyping of mice from 36 strains confirmed that while 20 strains (prototype strain BALB/c) possessed only one relative mass (Mr) for which typed C6A1 on IEF, the other 16 strains all possessed more than one C6 Mr form. Moreover, IEF analysis demonstrated additional polymorphic differences; among these 16 strains, 11 typed C6A1B1 like the prototype strain CBA, the AKR and RF/J strains typed C6A2B2, and the Japanese MOM strain as well as the C57BR/cdJ and C57L/J strains possessed two forms with IEF mobilities intermediate between C6A1B1 and C6A2B2. These will now be referred to as C6A3B3. Thus, a total of four different mouse C6 haplotypes have been identified. Testing C6 allotypes in a limited number of wild mice revealed that haplotypes found in inbred strains of Western or Eastern origin tend to reflect haplotypes of the wild mice from Europe or Japan, respectively.

Glucose transport across the blood-retinal barrier (BRB) was investigated in situ by means of a modification of the single-injection technique developed for studying transport across the blood-brain barrier. A retinal uptake index was calculated from the fractional extraction for 14C-glucose divided by the fractional extraction for the 3H2O reference. The use of the Eadie-Hofstee transformation to determine the Km of carrier-mediated transport revealed at least two transport systems for the unidirectional glucose flux across the BRB. Control data showed a relatively high-affinity system with a Km of 0.24 mM and a lower affinity system with a Km of 7.81 mM. Both these transport systems have been shown to be sensitive to phloretin but not to phloridzin or acetazolamide. It has also been shown that glucose transport is uneffected by Na+ or K+ concentrations but that calcium does have a significant effect on the lower affinity transport system for glucose. We have also demonstrated that the Km values of both transport systems are significantly increased by 2 months of streptozocin-induced diabetes.