What caused extinction of the Pleistocene megafauna of Sahul?

During the Pleistocene, Australia and New Guinea supported a rich assemblage of large vertebrates. Why these animals disappeared has been debated for more than a century and remains controversial. Previous synthetic reviews of this problem have typically focused heavily on particular types of evidence, such as the dating of extinction and human arrival, and have frequently ignored uncertainties and biases that can lead to misinterpretation of this evidence. Here, we review diverse evidence bearing on this issue and conclude that, although many knowledge gaps remain, multiple independent lines of evidence point to direct human impact as the most likely cause of extinction.

Secondary biochemical and morphological consequences in lysosomal storage diseases.

More than 50 hereditary lysosomal storage disorders (LSDs) are currently described. Most of these disorders are due to a deficiency of certain hydrolases/glycosidases and subsequent accumulation of nonhydrolyzable carbohydrate-containing compounds in lysosomes. Such accumulation causing hypertrophy of the lysosomal compartment is a characteristic feature of affected cells in LSDs. The investigation of biochemical and cellular parameters is of particular interest for understanding "life" of lysosomes in the normal state and in LSDs. This review highlights the wide spectrum of biochemical and morphological changes during developing LSDs that are extremely critical for many metabolic processes inside the various cells and tissues of affected persons. The data presented will help establish new complex strategies for metabolic correction of LSDs.

Cerebral Hirano-like bodies in an alpaca (Vicugna pacos): histologic and ultrastructural characterization.

A 14-year-old female alpaca (Vicugna pacos) was presented with a 1-week history of lethargy and anorexia and a 2-day history of recumbency, trembling, and hypothermia. There were no significant gross findings on postmortem examination. Hematoxylin and eosin-stained sections of brain demonstrated the presence of intracytoplasmic crystalline eosinophilic rod-shaped inclusions, mainly in the hippocampal pyramidal cells. Immunohistochemical staining for synuclein, tau protein, ubiquitin, and smooth muscle actin was negative. All inclusions were positive with phosphotungstic acid hematoxylin. Ultrastructurally, the inclusions were multilamellar and filamentous with longitudinal herringbone pattern and cross-sectional latticelike structure. The combination of hematoxylin and eosin appearance, special stains, immunostaining, and ultrastructural findings was consistent with Hirano-like bodies. The Hirano-like bodies were highly unlikely to be the cause of the neurologic signs experienced by this alpaca. To the authors' knowledge, this is the first report of spontaneous cerebral Hirano-like bodies in an alpaca.

Characterization of the withdrawal phase in a porcine donation after the cardiac death model.

Transplantation of donation after cardiac death (DCD) livers has higher rates of organ failure and complications, specifically ischemic biliary injuries. Reported large animal DCD models all employ active means to halt circulation, contrary to human DCD protocol. We report a DCD porcine model in which the animal passively progresses to cardiac death, thereby more closely mimicking human DCD scenario. Sixteen Yorkshire pigs (10 females, 6 males, 30-45 kg) had a mean time of 26:19 min ± 14:14 from withdrawal of ventilatory support (WVS) to circulatory arrest and 44:38 min ± 16:37 from WVS to electrical standstill. Cessation of hepatic flow (HF) occurred well before electrical standstill (22:15 min ± 10:09), previously not described in human or animal DCD. Histologically comparing livers from our DCD model demonstrated a dramatic increase in hepatocyte vacuolization, disorganization of endoplasmic reticulum, formation of mitochondrial inclusions and apoptosis compared with control specimens. Subtle changes were also evident in biliary epithelial cells (BEC). This results in severe cellular changes before reperfusion. Early histologic evidence suggests that there is severe hepatocyte and biliary cell disruption in our DCD model. Further research using this model may provide a deeper understanding of the pathophysiology of the DCD liver.

Favorable effect of VEGF gene transfer on ischemic peripheral neuropathy.

Ischemic peripheral neuropathy is a frequent, irreversible complication of lower extremity vascular insufficiency. We investigated whether ischemic peripheral neuropathy could be prevented and/or reversed by gene transfer of an endothelial cell mitogen designed to promote therapeutic angiogenesis. Intramuscular gene transfer of naked DNA encoding vascular endothelial growth factor (VEGF) simultaneously with induction of hindlimb ischemia in rabbits abrogated the substantial decrease in motor and sensory nerve parameters, and nerve function recovered promptly. When gene transfer was administered 10 days after induction of ischemia, nerve function was restored earlier and/or recovered faster than in untreated rabbits. These findings are due in part to enhanced hindlimb perfusion. In addition, however, the demonstration of functional VEGF receptor expression by Schwann cells indicates a direct effect of VEGF on neural integrity as well. These findings thus constitute a new paradigm for the treatment of ischemic peripheral neuropathy.

Pathology of GM2 gangliosidosis in Jacob sheep.

The G(M2) gangliosidoses are a group of lysosomal storage diseases caused by defects in the genes coding for the enzyme hexosaminidase or the G(M2) activator protein. Four Jacob sheep from the same farm were examined over a 3-year period for a progressive neurologic disease. Two lambs were 6-month-old intact males and 2 were 8-month-old females. Clinical findings included ataxia in all 4 limbs, proprioceptive deficits, and cortical blindness. At necropsy, the nervous system appeared grossly normal. Histologically, most neurons within the brain, spinal cord, and peripheral ganglia were enlarged, and the cytoplasm was distended by foamy to granular material that stained positively with Luxol fast blue and Sudan black B stains. Other neuropathologic findings included widespread astrocytosis, microgliosis, and scattered spheroids. Electron microscopy revealed membranous cytoplasmic bodies within the cytoplasm of neurons. Biochemical and molecular genetic studies confirmed the diagnosis of G(M2) gangliosidosis. This form of G(M2) gangliosidosis in Jacob sheep is very similar to human Tay-Sachs disease and is potentially a useful animal model.

Biology of Giardia lamblia. Detection of N-acetyl-D-glucosamine as the only surface saccharide moiety and identification of two distinct subsets of trophozoites by lectin binding.

Lectins and glycosidases of known sugar specificity were used as probes to analyze the surface carbohydrate moieties of G. lamblia trophozoites and in particular to determine whether chitin or oligomeric D-GlcNAc is present in the trophozoite form of the parasite as well as on the cyst. Of 13 lectins with varying sugar specificity, only D-GlcNAc-specific lectins bound specifically to the trophozoite surface as determined by light microscopy and EM. A striking finding was the identification of two distinct subsets of trophozoites, distinguished by reactivity with WGA and detected by light microscopy and EM as well as by flow cytometry. Unlike the cyst wall, the trophozoite D-GlcNAc residues were resistant to chitinase treatment. In contrast N-acetyl-beta-D-glucosaminidase abolished WGA binding suggesting that the lectin binds to terminal beta-linked D-GlcNAc residues. These residues were identified as being present on surface glycoproteins by Western blotting of parasite membrane proteins using WGA as a probe. This study identifies D-GlcNAc as the only saccharide moiety detectable by lectin binding on the surface of G. lamblia trophozoites and demonstrates that in contrast to the cyst, chitin is not present in the trophozoite. In addition two distinct subsets of trophozoites were identified based on reactivity with WGA and may represent varying stages of differentiation from trophozoite to cyst.

Freeze-fracture of monolayer cultures.

This paper describes a simple method for the freeze-fracturing of cells in monolayers or multi-layer tissue cultures. The method produces high quality replicas and is applicable to the study of virtually any tissue culture or organ culture system. It uses standard materials and equipment for both tissue culture and freeze-fracturing.

Cope's rule and the dynamics of body mass evolution in North American fossil mammals.

Body mass estimates for 1534 North American fossil mammal species show that new species are on average 9.1% larger than older species in the same genera. This within-lineage effect is not a sampling bias. It persists throughout the Cenozoic, accounting for the gradual overall increase in average mass (Cope's rule). The effect is stronger for larger mammals, being near zero for small mammals. This variation partially explains the unwavering lower size limit and the gradually expanding mid-sized gap, but not the sudden large increase in the upper size limit, at the Cretaceous-Tertiary boundary.

A multispecies overkill simulation of the end-Pleistocene megafaunal mass extinction.

A computer simulation of North American end-Pleistocene human and large herbivore population dynamics correctly predicts the extinction or survival of 32 out of 41 prey species. Slow human population growth rates, random hunting, and low maximum hunting effort are assumed; additional parameters are based on published values. Predictions are close to observed values for overall extinction rates, human population densities, game consumption rates, and the temporal overlap of humans and extinct species. Results are robust to variation in unconstrained parameters. This fully mechanistic model accounts for megafaunal extinction without invoking climate change and secondary ecological effects.

Isolation of T-lymphotropic retrovirus related to HTLV-III/LAV from wild-caught African green monkeys.

Present evidence suggests that the acquired immune deficiency syndrome (AIDS) emerged in Central Africa as a new disease in recent decades. This disease has recently approached epidemic proportions in many parts of the world. The etiologic agent of AIDS is believed to be the virus HTLV-III/LAV, which has been proposed as having originated from a recent simian-human transmission in Africa. This report describes the isolation of a designated STLV-IIIAGM retrovirus closely related to HTLV-III/LAV from seven healthy wild-caught African Green monkeys (Cercopithecus aethiops) that showed the presence of antibodies designated STLV-IIIAGM. In vitro growth characteristics, ultrastructural morphology, and major proteins of 160,000 kilodaltons (kD), 120 kD, 55 kD, and 24 kD are similar to and cross-reactive with the analogous antigens of HTLV-III/LAV. The use of these serologic markers in the detection of STLV-IIIAGM-infected monkeys may be important in assuring the continued safety of a variety of biologic reagents that are derived from these primate species. The existence of a retrovirus closely related to HTLV-III/LAV that naturally infects an African nonhuman primate in the apparent absence of disease may provide a unique model for the study of human AIDS and the development of an effective vaccine.

Neurovisceral and skeletal GM1-gangliosidosis in dogs with beta-galactosidase deficiency.

Beta-galactosidase-deficient siblings in two litters of English springer spaniel puppies showed a progressive neurological impairment, dwarfism, orbital hypertelorism, and dysostosis multiplex. An excess of GM1-ganglioside was found in the brain. Three abnormal oligosaccharides were present in samples of urine, brain, liver, and cartilage. Light microscopy of selected tissue specimens revealed cytoplasmic vacuoles in neurons, circulating blood cells, macrophages, and chondrocytes. Ultrastructural studies demonstrated that these membrane-bound vacuoles were of two types--one containing lamellated membranes and the other, finely granular material. These clinical and pathological findings are similar to those observed in human patients affected by the infantile form of GM1-gangliosidosis.

Spontaneous appearance of Tay-Sachs disease in an animal model.

Tay-Sachs disease (TSD) is a progressive neurodegenerative disorder due to an autosomal recessively inherited deficiency of beta-hexosaminidase A (Hex A). Deficiency of Hex A in TSD is caused by a defect of the alpha-subunit resulting from mutations of the HEXA gene. To date, there is no effective treatment for TSD. Animal models of genetic diseases, similar to those known to exist in humans, are valuable and essential research tools for the study of potentially effective therapies. However, there is no ideal animal model of TSD available for use in therapeutic trials. In the present study, we report an animal model (American flamingo; Phoenicopterus ruber) of TSD with Hex A deficiency occurring spontaneously in nature, with accumulation of G(M2)-ganglioside, deficiency of Hex A enzymatic activity, and a homozygous P469L mutation in exon 12 of the hexa gene. In addition, we have isolated the full-length cDNA sequence of the flamingo, which consists of 1581 nucleotides encoding a protein of 527 amino acids. Its coding sequence indicates approximately 71% identity at the nucleotide level and about 72.5% identity at the amino acid level with the encoding region of the human HEXA gene. This animal model, with many of the same features as TSD in humans, could represent a valuable resource for investigating therapy of TSD.

A neuraminidase from Trypanosoma cruzi removes sialic acid from the surface of mammalian myocardial and endothelial cells.

Trypanosoma cruzi causes Chagasic heart disease, a major public health problem in Latin America. The mechanism of interaction of this protozooan parasite with host cells is poorly understood. We recently found that the infective trypomastigote form a T. cruzi exhibits neuraminidase activity and can desialylate mammalian erythrocytes. However, it is not known if T. cruzi can also modify the surfaces of cardiovascular cells that are directly involved in the most important clinical manifestations of this disease. Accordingly, this study determined whether T. cruzi can remove sialic acid from cultured rat myocardial or human vascular endothelial cells. Sialic acid was labeled metabolically with the precursor 3H-N-acetyl-D-mannosamine. Soluble neuraminidase, isolated from intact T. cruzi trypomastigotes, caused significant release of labeled material from myocardial cells (e.g., 2,174 +/- 27 dpm/h vs. spontaneous release of 306 +/- 30 dpm/h, n = 4, P less than 0.001). Chromatographic analysis showed that the bulk of the radioactivity released by T. cruzi neuraminidase was sialic acid. Intact T. cruzi trypomastigotes also released sialic acid from metabolically labeled myocardial cells in a concentration-dependent manner. In contrast, a noninfective form of T. cruzi, the amastigote, did not desialylate these cells. Galactose oxidase labeling demonstrated newly desialylated glycoproteins on the surface of myocardial cells treated with T. cruzi neuraminidase. Desialylation of myocardial cells was confirmed histochemically by the appearance of binding sites for peanut agglutinin, a lectin that binds to complex oligosaccharide moieties after removal of the terminal sialyl residue. T. cruzi neuraminidase also removed sialic acid from adult human saphenous vein endothelial cells, as determined by both histochemical and metabolic labeling studies. Thus, infective forms of T. cruzi can chemically modify the surfaces of myocardial and vascular endothelial cells by desialylation. This alteration may play a role in the initial interaction of this parasite with these important target cells of the host cardiovascular system.

The role of the eae gene of enterohemorrhagic Escherichia coli in intimate attachment in vitro and in a porcine model.

The eaeA gene of enteropathogenic Escherichia coli (EPEC) is necessary for intimate attachment to epithelial cells in vitro. Enterohemorrhagic E. coli (EHEC) strains also possess an eae gene and are capable of intimate attachment and microvillus effacement in vitro and in animal models. To assess the role of the EHEC eae gene in intimate attachment, we constructed an eae deletion/insertion mutation in wild-type EHEC O157:H7 strain 86-24 by using linear electroporation of a recombinant allele. The mutant obtained was deficient in inducing f-actin accumulation in HEp-2 cells and was incapable of attaching intimately to colonic epithelial cells in a newborn piglet model of infection. Intimate attachment in vivo was restored when the EHEC eae gene or the eaeA gene of EPEC was introduced into the mutant on a plasmid. These results indicate that the eae gene is necessary for intimate attachment of EHEC in vivo. In addition, the complementation achieved by the EPEC locus indicates that the eae gene of EHEC and the eaeA gene of EPEC are functionally homologous.

Unusual cell junctional complexes in canine mammary adenoacanthomas.

Unusual cell junctional complexes were described in spontaneously arising adenoacanthomas of canine mammary glands. These junctional complexes were a manifestation of bidirectional differentiation of tumor cell membranes.

Desmosome ultrastructure and biological behavior of chemical carcinogen-induced urinary bladder carcinomas.

In the quantitative electron microscopic study, we examined the relationship of desmosomes to tumor invasiveness in chemical carcinogen (N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide)-induced urinary bladder transitional cell carcinomas in the Fischer rat. The number of a desmosomes per unit area of plasma membrane was significantly reduced in carcinomas. However, the percentage of cell surface area occupied by desmosomes was greater in carcinomas than in controls. This was accounted for on the basis of increases in desmosomal size, which result from squamous differentiation within the tumors. Areas of transitional cell differentiation and squamous differentiation demonstrated an equal capacity for invasiveness. Desmosomes were abundant in invading nests of tumor cells. These findings cast doubt on the validity of the concept of decreased intercellular adhesion as a prerequisite for tumor invasion, since strong interadhesion is probably a function of the area occupied by the intercellular junctions.

Heterogeneous distribution of filipin-sterol complexes in nuclear membranes.

Filipin, a sterol-specific polyene antibiotic, has been shown by electron microscopy to form complexes in membranes of mouse urinary bladder cells. Following instillation of a glutaraldehyde-filipin-dimethylsulfoxide solution into the bladder lumen, filipin-cholesterol complexes appear as membrane corrugations in thin sections and as 20-25 nm protuberances and depressions on PF and EF faces in freeze-fracture replicas. The complexes are observed in plasmalemma, Golgi membrane, rough endoplasmic reticulum and nuclear membrane of five different cell types (urothelial, endothelial, mesothelial, smooth muscle and fibroblasts). In the present report, we direct particular attention to the localization of numerous filipin-cholesterol complexes present in the nuclear envelopes of these cells. Our results suggest that enrichment of cell membranes with cholesterol occurs at an earlier stage in the flow-differentiation process than previously suspected. In addition, the unequal distribution of complexes in favor of the outer nuclear membrane suggests that it has a higher cholesterol content than the inner membrane.

Correction of acid beta-galactosidase deficiency in GM1 gangliosidosis human fibroblasts by retrovirus vector-mediated gene transfer: higher efficiency of release and cross-correction by the murine enzyme.

Mutations in the lysosomal acid beta-galactosidase (EC 3.2.1.23) underlie two different disorders: GM1 gangliosidosis, which involves the nervous system and visceral organs to varying extents, and Morquio's syndrome type B (Morquio B disease), which is a skeletal-connective tissue disease without any CNS symptoms. This article shows that transduction of human GM1 gangliosidosis fibroblasts with retrovirus vectors encoding the human acid beta-galactosidase cDNA leads to complete correction of the enzymatic deficiency. The newly synthesized enzyme is correctly processed and targeted to the lysosomes in transduced cells. Cross-correction experiments using retrovirus-modified cells as enzyme donors showed, however, that the human enzyme is transferred at low efficiencies. Experiments using a different retrovirus vector carrying the human cDNA confirmed this observation. Transduction of human GM1 fibroblasts and mouse NIH 3T3 cells with a retrovirus vector encoding the mouse beta-galactosidase cDNA resulted in high levels of enzymatic activity. Furthermore, the mouse enzyme was found to be transferred to human cells at high efficiency. Enzyme activity measurements in medium conditioned by genetically modified cells suggest that the human beta-galactosidase enzyme is less efficiently released to the extracellular space than its mouse counterpart. This study suggests that lysosomal enzymes, contrary to the generalized perception in the field of gene therapy, may differ significantly in their properties and provides insights for design of future gene therapy interventions in acid beta-galactosidase deficiency.

Mucolipidosis IV: morphology and histochemistry of an autopsy case.

Mucolipidosis Type IV is a rare, autosomal recessive disorder characterized by corneal opacification, mental retardation, and delayed motor milestones. Whereas lysosomal storage material has been demonstrated in biopsied tissues and leukocytes, the complete autopsy pathology, including neuropathology, is unknown. The metabolic defect remains speculative. We report the general and neuropathologic findings of the only known autopsy. In the central nervous system, neuronal loss in the cerebral cortex, basal ganglia, deep cerebellar nuclei, and brainstem nuclei was marked by astrocytosis; the cytoplasm of residual neurons had brown granules. These granules were positive with periodic acid-Schiff, Concanavalia ensiformis, and Sudan black, but not with Luxol-fast blue. Ultrastructurally, neurons contained lysosomes laden with osmiophilic, amorphous and granular material, and few lamellated membrane structures. Hepatocytes, epithelia, endothelia, chondrocytes, and tissue macrophages also stained positively with Datura stramonium and Ricinus communis-I agglutinins, with renal glomeruli also staining with peanut agglutinin; most non-neural cells contained osmiophilic granules on toluidine blue-stained, plastic embedded sections, corresponding to lamellated membrane structures. These findings complement the previously reported ocular morphology and brain and liver biochemistry performed in the same patient, and suggest that the storage material in neurons differs from that in non-neural cells. Furthermore, the underlying defect is not likely to be a deficiency of a single enzyme (i.e. a lysosomal hydrolase).