Physiological variability in yearling alligators: clutch differences at rest and during activity.

The adult phenotype of an organism is the result of its genotype, the environment, and the interaction between the two. Assessing the relative contribution of these factors to the final adult phenotype continues to occupy researchers. Studies have shown clutch effects early in development but few have investigated the persistence of clutch effects on a longer time scale. Five clutches of American alligators were reared for 1 year in a common environment then assessed for the presence of clutch effects as they related to morphological and physiological characteristics. After 1 year, significant clutch effects were evident in all size related variables despite open access to food. Additionally, lung and liver masses remained different between clutches after animal mass was taken into account. Although clutch had no effect on resting heart rate, it significantly contributed to mean arterial pressure. During swimming and exhaustive exercise, the resulting respiratory and metabolic acidoses were strongly dependent on clutch. Therefore, while the environment can have significant influences on the American alligator from hatching to death, the measureable contribution of genetics to the morphology and physiology of the organism remains evident, even after 1 year of common rearing conditions. It behooves researchers to acknowledge and control for clutch effects when designing experiments.

Expanding the PET radioisotope universe utilizing solid targets on small medical cyclotrons.

Molecular imaging with medical radioisotopes enables the minimally-invasive monitoring of aberrant biochemical, cellular and tissue-level processes in living subjects. The approach requires the administration of radiotracers composed of radioisotopes attached to bioactive molecules, the pairing of which considers several aspects of the radioisotope in addition to the biological behavior of the targeting molecule to which it is attached. With the advent of modern cellular and biochemical techniques, there has been a virtual explosion in potential disease recognition antigens as well as targeting moieties, which has subsequently opened new applications for a host of emerging radioisotopes with well-matched properties. Additionally, the global radioisotope production landscape has changed rapidly, with reactor-based production and its long-defined, large-scale centralized manufacturing and distribution paradigm shifting to include the manufacture and distribution of many radioisotopes via a worldwide fleet of cyclotrons now in operation. Cyclotron-based radioisotope production has become more prevalent given the commercial availability of instruments, coupled with the introduction of new target hardware, process automation and target manufacturing methods. These advances enable sustained, higher-power irradiation of solid targets that allow hospital-based radiopharmacies to produce a suite of radioisotopes that drive research, clinical trials, and ultimately clinical care. Over the years, several different radioisotopes have been investigated and/or selected for radiolabeling due to favorable decay characteristics (i.e. a suitable half-life, high probability of positron decay, etc.), well-elucidated chemistry, and a feasible production framework. However, longer-lived radioisotopes have surged in popularity given recent regulatory approvals and incorporation of radiopharmaceuticals into patient management within the medical community. This review focuses on the applications, nuclear properties, and production and purification methods for some of the most frequently used/emerging positron-emitting, solid-target-produced radioisotopes that can be manufactured using small-to-medium size cyclotrons (≤24 MeV).

Contribution of gular pumping to lung ventilation in monitor lizards.

A controversial hypothesis has proposed that lizards are subject to a speed-dependent axial constraint that prevents effective lung ventilation during moderate- and high-speed locomotion. This hypothesis has been challenged by results demonstrating that monitor lizards (genus Varanus) experience no axial constraint. Evidence presented here shows that, during locomotion, varanids use a positive pressure gular pump to assist lung ventilation. Disabling the gular pump reveals that the axial constraint is present in varanids but it is masked by gular pumping under normal conditions. These findings support the prediction that the axial constraint may be found in other tetrapods that breathe by costal aspiration and locomote with a lateral undulatory gait.

MUC-2 human small intestinal mucin gene structure. Repeated arrays and polymorphism.

MUC-2, the first described intestinal mucin gene, has become important as a prototype for secreted mucins in several organ systems. However, little is known about its protein backbone structure and hence its role in diseases such as colon cancer, ulcerative colitis, and cystic fibrosis, which are known to have mucin abnormalities. Studies in this manuscript show that MUC-2 contains two distinct regions with a high degree of internal homology, but the two regions bear no significant homology to each other. Region 1 consists mostly of 48-bp repeats which are interrupted in places by 21-24-bp segments. Several of these interrupting sequences show similarity to each other, creating larger composite repeat units. Region 1 has no length polymorphisms. Region 2 is composed of 69-bp tandem repeats arranged in an uninterrupted array of up to 115 individual units. Southern analysis of genomic DNA samples using TaqI and HinfI reveals both length and sequence polymorphisms which occur within region 2. The sequence polymorphisms have different ethnic distributions, while the length polymorphisms are due to variable numbers of tandem repeats.

Molecular cloning of complementary DNAs encoding alkaline phosphatase in human colon cancer cells.

We have isolated a set of complementary DNA (cDNA) clones that together encode the alkaline phosphatase of human colon cancer LS174T cells. These clones include two cDNAs isolated from a conventionally prepared oligodeoxythymidylate-primed lambda ZAP cDNA library and three cDNA clones prepared by using the polymerase chain reaction. The deduced amino acid sequence of the alkaline phosphatase primary transcript contains 532 amino acids. This enzyme is similar to, but not identical with, placental alkaline phosphatase (PLAP); it exhibits 12-19 amino acid substitutions when compared to the various alleles of PLAP. Also, it is similar to PLAP in that it is apparently attached to the cell membrane by a phosphatidylinositol-containing anchor as judged by the ability of phosphatidylinositol-specific phospholipase C to release it from membranes. It is different from PLAP however, in terms of its signal sequence which only contains 19 amino acids as compared to 22 for PLAP. Moreover, the 3'-untranslated region of the LS174T cell alkaline phosphatase message diverges considerably from the PLAP message. The LS174T cell alkaline phosphatase cDNAs are actually much more similar to the "germ cell" alkaline phosphatase gene than they are to PLAP. Only 7 amino acid substitutions exist between the LS174T cell enzyme and the alkaline phosphatase encoded by the germ cell alkaline phosphatase genomic DNA clone isolated by Millan and Manes (Proc. Natl. Acad. Sci. USA, 85: 3024-3028, 1988). Furthermore, the 3'-untranslated region of the LS174T cell alkaline phosphatase message is very similar to the sequence immediately downstream of the coding region of the germ cell alkaline phosphatase genomic DNA clone. Thus, these results indicate that this colon cancer cell alkaline phosphatase is likely to represent an allelic variant encoded at the germ cell alkaline phosphatase locus.

Mouse intestinal goblet cells expressing SV40 T antigen directed by the MUC2 mucin gene promoter undergo apoptosis upon migration to the villi.

Mucinous colorectal cancers exhibit a characteristic set of molecular genetic alterations and may be derived from progenitor cells committed to the goblet cell lineage. Previously, we demonstrated that the MUC2 mucin gene promoter drives transgene reporter expression with high specificity in small intestinal goblet cells of transgenic mice. On the basis of these experiments, we reasoned that the MUC2 promoter could be used to drive SV40 T antigen (Tag) expression in the same cell type, decoupling them from their normal antiproliferative controls. A line of mice was established (MUCTag6) that expressed Tag in intestinal goblet cells as determined by RNA blot and immunohistochemical analysis. These goblet cells were markedly involuted however, most notably in the villi. Endogenous intestinal MUC2 message levels were reduced to about one third the normal level in these mice. However, absorptive cell lineage markers were comparable with nontransgenics. Bromodeoxyuridine-positive S-phase cells are limited to crypts in nontransgenic intestine but are present in both crypts and villi in MUCTag6. In contrast, mitotic cells were not present in the villi, indicating that MUCTag6 villi goblet cells do not progress into M phase. Apoptotic cells positive for terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling were increased more than fourfold in MUCTag6 villi (P < 0.0001), and apoptotic goblet cells were evident. Electron microscopic examination of MUCTag6 intestinal villi revealed the presence of degraded cell remnants containing mucin goblets together with other cell debris, further indicating apoptosis of the goblet cell lineage. Thus, the expression of Tag in intestinal goblet cells releases them from normal antiproliferative controls, causing their inappropriate entry into S phase even after they transverse the crypt/villus junction. They do not, however, progress to M phase. Instead, they undergo apoptosis with a high degree of efficiency in S or G(2) phase. These experiments demonstrate that apoptosis effectively blocks inappropriate goblet cell proliferation in the intestine, supporting its proposed role as an antineoplastic mechanism.

The effect of meal composition on specific dynamic action in burmese pythons (Python molurus).

We quantified the specific dynamic action (SDA) resulting from the ingestion of various meal types in Burmese pythons (Python molurus) at 30 degrees C. Each snake was fed a series of experimental meals consisting of amino acid mixtures, simple proteins, simple or complex carbohydrates, or lipids as well as meals of whole animal tissue (chicken breast, beef suet, and mouse). Rates of oxygen consumption were measured for approximately 4 d after feeding, and the increment above standard metabolic rate was determined and compared to energy content of the meals. While food type (protein, carbohydrate, and lipid) had a general influence, SDA was highly dependent on meal composition (i.e., amino acid composition and carbohydrate structure). For chicken breast and simple carbohydrates, the SDA coefficient was approximately one-third the energetic content of the meal. Lard, suet, cellulose, and starch were not digested and did not produce measurable SDA. We conclude that the cost of de novo protein synthesis is an important component of SDA after ingestion of protein meals because (1) simple proteins, such as gelatin and collagen, did not stimulate levels of SDA attained after consumption of complete protein, (2) incomplete mixtures of amino acids failed to elicit the SDA of a complete mixture, and (3) the inhibition of de novo protein synthesis with the drug cycloheximide caused a more than 70% decrease in SDA. Stomach distension and mechanical digestion of intact prey did not cause measurable SDA.

Isolation and biological activity of a novel kinin ([Thr6] bradykinin) from the turtle, Pseudemys scripta.

Incubation of plasma from the red-eared turtle with glass beads in the presence of the kininase inhibitor 1,10-phenanthroline resulted in activation of the kallikrein-kinin system and generation of bradykinin-like immunoreactivity. The immunoreactive material comprised a single molecular form that was purified to homogeneity by reverse phase HPLC. The primary structure of the peptide was determined by automated Edman degradation and fast atom bombardment mass spectrometry. The amino acid sequence of the turtle kinin Arg-Pro-Pro-Gly-Phe-Thr-Pro-Phe-Arg contains the substitution Thr for Ser at position 6 compared with mammalian bradykinin. [Thr6] bradykinin was synthesized using solid phase methodology, and bolus injections of the peptide into the left atrium of the anaesthetized turtle produced rapid vasodilation. A dose-dependent increase in blood flow in the left aortic arch was accompanied by a decrease in peripheral vascular resistance, so that systemic blood pressure did not change. The data suggest that the kallikrein-kinin system may play an important physiological role in the regulation of cardiovascular function in reptiles.

Analysis of dipeptidyl peptidase IV gene regulation in transgenic mice: DNA elements sufficient for promoter activity in the kidney, but not the intestine, reside on the proximal portion of the gene 5'-flanking region.

The dipeptidyl peptidase IV (DPPIV) gene encodes a brush border membrane exopeptidase that is expressed in a tissue-restricted fashion. To examine the regulation of DPPIV transcription in various tissues in vivo, we examined the expression of DPPIV 5'-flanking region (promoter)-human growth hormone reporter constructs in transgenic mice. These mice exhibited cell-type specific reporter expression in kidney. Surprisingly, however, only very low to non-detectable levels of reporter were found in small intestine. These results indicate that DNA elements sufficient for DPPIV expression in kidney, but not intestine, reside in the 5'-flanking region of the gene.

An experimental test of the thermoregulatory hypothesis for the evolution of endothermy.

The thermoregulatory hypothesis proposes that endothermy in mammals and birds evolved as a thermoregulatory mechanism per se and that natural selection operated directly to increase body temperature and thermal stability through increments in resting metabolic rate. We experimentally tested this hypothesis by measuring the thermoregulatory consequences of increased metabolic rate in resting lizards (Varanus exanthematicus). A large metabolic increment was induced by feeding the animals and consequent changes in metabolic rate and body temperature were monitored. Although metabolic rate tripled at 32 degrees C and quadrupled at 35 degrees C, body temperature rose only about 0.5 degrees C. The rate of decline of body temperature in a colder environment did not decrease as metabolic rate increased. Thus, increasing the visceral metabolic rate of this ectothermic lizard established neither consequential endothermy nor homeothermy. These results are inconsistent with a thermoregulatory explanation for the evolution of endothermy.

Absolute phasic blood flow measurement in the brain using digital subtraction angiography.

RATIONALE AND OBJECTIVES: In this study, an angiographic method using first-pass distribution analysis was used for quantification of phasic volumetric blood flow. Studies were conducted in an angiographic perfusion phantom and in an animal model (rabbit) after intraarterial injection of contrast material. METHODS: Digital subtraction angiography and first-pass distribution algorithm were used to assess the absolute volumetric flow rates. The method, based on the conservation of contrast material in successive angiographic images, uses the videodensitometric information in the arterial bed. Measurements were made by summing the pixel values in the region of interest, covering the whole perfusion bed. A contrast pass curve was generated for a sequence of images to obtain instantaneous volumetric flow rates. RESULTS: Instantaneous and mean absolute volumetric flow measurements made in the angiographic perfusion phantom and the common carotid artery of the animal models correlated well with validation measurements made using ultrasound flowprobes. The measured (M) and known (K) flow rates in the carotid artery were related by M = 0.87 K + 2.50 mL/minute (r = 0.96, standard error of the estimate = 3.79 mL/minute, n = 25) and M = 0.92 K - 1.00 mL/minute (r = 0.98, standard error of the estimate = 4.04 mL/minute, n = 38) using the videodensitometric and entrance vessel calibration techniques, respectively. Conclusion. Results demonstrate the potential use of the first-pass distribution method in conjunction with digital subtraction angiography for measuring phasic arterial blood flow in vivo.

Isolation and structural characterization of insulin, glucagon and somatostatin from the turtle, Pseudemys scripta.

The chelonians occupy an important position in phylogeny representing a very early branching from the ancestral reptile stock. Hormonal polypeptides in an extract of the pancreas of the red-eared turtle were purified to homogeneity by reversed phase HPLC and their primary structures were determined. Turtle insulin is identical to chicken insulin. Turtle glucagon differs from chicken glucagon by the substitution of a serine by a threonine residue at position 16 and from mammalian glucagon by an additional substitution of an asparagine by a serine residue at position 28. Turtle pancreatic somatostatin is identical to mammalian somatostatin-14. The crocodilians are phylogenetically much closer to the birds than are the chelonians. Alligator insulin, however, contains three amino acid substitutions relative to chicken insulin. Thus, caution is required when inferring phylogenetic relationships based upon a comparison of amino acid sequences of homologous peptides.

Effect of sodium butyrate, a differentiating agent, on cell surface glycoconjugates of a human pancreatic cell line.

The effects of sodium butyrate, a differentiating agent, on growth properties and glycoconjugates of a human pancreatic carcinoma cell line (CAPAN-1) were studied. Butyrate caused marked changes of in vitro growth properties including prolongation of doubling time and greatly reduced colony forming efficiency in soft agar. Cell surface labeling revealed significant alterations in proteins and glycoproteins after treatment with sodium butyrate (appearance of glycoproteins of molecular weight 250,000, 220,000, and 70,000; reduction of glycoproteins of 164,000, 148,000, 110,000, and 66,800 sizes; increases in proteins/glycoproteins of 85,000 and 78,000). Metabolic labeling of the cells with [3H]fucose or [3H]galactose revealed that sodium butyrate treatment caused a marked reduction in the fucose-containing neutral glycolipids with six or more carbohydrate side chains and an increase in [3H]galactose-labeled neutral glycolipids, particularly GL-3a, GL-4a, and GL-5a. There was marked reduction in the labeling of a ganglioside with mobility similar to that of GM4 and of sulfogalactosylceramide. An increase in a ganglioside with mobility above GM1 was also caused by butyrate. These data indicate that sodium butyrate may be useful in the identification of differentiation or malignancy-associated glycoconjugate markers of human pancreatic cells.

Evidence that neuromedin U may regulate gut motility in reptiles but not in mammals.

Neuromedin U-8 induced a monophasic and concentration-dependent contraction of intact small intestine from the turtle, Pseudemys scripta, whereas the peptide had no effect upon the motility of rat and guinea pig gut. The maximum response produced by neuromedin U-8 was 56% of that produced by acetylcholine and 62% of that produced by potassium chloride. The potency and maximum response to neuromedin U-8 were unaffected by tetrodotoxin and atropine. The data suggest that neuromedin U may play a role in regulation of gut motility in lower vertebrates but not in mammals.

Voice stress evaluators and lie detection.

Proponents of vocal stress analysis systems argue that they are able to detect spoken deception by analysis of "stress" in the voice signal. Presumably, they do so by examining traces made by laryngeal microtremors which, they claim exist in the voice, are associated with stress, and ultimately are associated with lying. However, most research that seeks to identify the relationships between microtremors and laryngeal function has produced negative results, and data on the ability of voice analyzers to detect stress from speech--or to identify spoken deception--have been negative or "mixed" in nature. Since perspectives based on available results leave a number of questions unanswered, a series of experiments has been undertaken. The first was focused on the basic acoustic/temporal correlates of stress in voice (the subject of an earlier report), the second on examination of stress by commercial voice analyzers, and the third on the detection of relatively high-risk lies by this same type of voice analysis procedure. It was found that correct stress/nonstress identifications occurred only at chance levels; the lie/nonlie identification scores were quite similar with professional "examiners" performing at about the same level of accuracy as other auditors. The following review is divided into two parts: a history of the controversy and a presentation of the two cited experiments.

Temperature resistance of Salmonella in low-water activity whey protein powder as influenced by salt content.

Salmonella can survive in low-water activity (a(w)) foods for long periods of time. Water activity and the presence of solutes may affect its survival during heating. Low-a(w) products that contain sodium levels above 0.1 % (wt/wt) and that have been involved in major Salmonella outbreaks include peanut products and salty snacks. Reduced a(w) protects against thermal inactivation. There is conflicting information regarding the role of salt. The aim of this study was to determine whether NaCl influences the survival of Salmonella in low-a(w) whey protein powder independent of a(w) at 70 and 80 °C. Whey protein powders of differing NaCl concentrations (0, 8, and 17 % [wt/wt]) were equilibrated to target a(w) levels 0.23, 0.33, and 0.58. Powders were inoculated with Salmonella, vacuum sealed, and stored at 70 and 80 °C for 48 h. Cells were recovered on nonselective differential media. Survival data were fit with the Weibull model, and first decimal reduction times (δ) (measured in minutes) and shape factor values (ß) were estimated. The influence of temperature, a(w), and salinity on Weibull model parameters (δ and ß) was analyzed using multiple linear regression. Results showed that a(w) significantly influenced the survival of Salmonella at both temperatures, increasing resistance at decreasing a(w). Sodium chloride did not provide additional protection or inactivation of Salmonella at any temperature beyond that attributed to a(w). The Weibull model described the survival kinetics of Salmonella well, with R2 adj and root mean square error values ranging from 0.59 to 0.97 and 0.27 to 1.07, respectively. Temperature and a(w) influenced δ values (P < 0.05), whereas no significant differences were found between 70 and 80 °C among the different salt concentrations (P > 0.05). ß values were not significantly influenced by temperature, a(w), or % NaCl (P > 0.05). This study indicates that information on salt content in food may not help improve predictions on the inactivation kinetics of Salmonella in low-a(w) protein systems within the a(w) levels and temperatures studied.

Relative survival of four serotypes of Salmonella enterica in low-water activity whey protein powder held at 36 and 70°C at various water activity levels.

Salmonella enterica is not able to grow at water activity (aw) levels below 0.94, but it can survive in low-aw foods for long periods of time. Temperature, aw, substrate, and serotype affect its persistence. The aim of this study was to evaluate the influence of temperature and aw on the relative persistence among four serotypes of Salmonella enterica in low-aw whey protein powder. Whey protein powder was equilibrated to aws 0.18 ± 0.02 and 0.54 ± 0.03, inoculated with a cocktail of Salmonella serovars (Agona, Tennessee, Montevideo, and Typhimurium), vacuum sealed, and stored at 36°C for 6 months and at 70°C for 48 h. Presumptive Salmonella colonies (30 to 32) were randomly picked from each plate at the end of each survival study. PCR multiplex serotyping was used to identify the isolates. A multinomial mixed logistic model with Salmonella Tennessee as a reference was used to test for significant differences in frequency distribution of the surviving serotypes. Salmonella Tennessee and Salmonella Agona were the most prevalent surviving serotypes, followed in decreasing order by Salmonella Montevideo and Salmonella Typhimurium. Statistical analysis indicated that temperature (P = 0.003) and aw (P = 0.012) influenced the relative prevalence of the Salmonella serotypes. If other environmental conditions are equal, Salmonella Tennessee is better able to survive than Salmonella Montevideo and Salmonella Typhimurium at higher temperatures and higher aw levels in low-aw whey protein powder held at 36 and 70°C. The relative prevalence of Salmonella Agona to Salmonella Tennessee did not change with increasing temperature (P = 0.211) or aw (P = 0.453). These results should be considered in risk assessment and when developing predictive models for survival of Salmonella in low-aw foods.

Comparative cardiovascular physiology: future trends, opportunities and challenges.

The inaugural Kjell Johansen Lecture in the Zoophysiology Department of Aarhus University (Aarhus, Denmark) afforded the opportunity for a focused workshop comprising comparative cardiovascular physiologists to ponder some of the key unanswered questions in the field. Discussions were centred around three themes. The first considered function of the vertebrate heart in its various forms in extant vertebrates, with particular focus on the role of intracardiac shunts, the trabecular ('spongy') nature of the ventricle in many vertebrates, coronary blood supply and the building plan of the heart as revealed by molecular approaches. The second theme involved the key unanswered questions in the control of the cardiovascular system, emphasizing autonomic control, hypoxic vasoconstriction and developmental plasticity in cardiovascular control. The final theme involved poorly understood aspects of the interaction of the cardiovascular system with the lymphatic, renal and digestive systems. Having posed key questions around these three themes, it is increasingly clear that an abundance of new analytical tools and approaches will allow us to learn much about vertebrate cardiovascular systems in the coming years.

The energetic consequences of dietary specialization in populations of the garter snake, Thamnophis elegans.

We investigated the intraspecific variation in digestive energetics between dietary specialist and generalist populations of the Western Terrestrial garter snake (Thamnophis elegans) in northern California. Coastal populations have a specialized diet of slugs and inland populations have a generalized diet of fish, anurans, mice and leeches. The difference in prey preference between the two populations is congenital, heritable and ontogenetically stable. To examine energetic specializations and trade-offs in these populations, we measured the net assimilation efficiency of each snake population on both slug (Ariolimax columbianus) and fish (Rhinichthys osculus) diets. The net assimilation efficiency was measured during digestion of a meal and continued until metabolic rate re-attained prefeeding levels. Coastal snakes were able to utilize 62% more of the ingested energy towards production from slug diets through both increased assimilation of nutrients and reduced digestive costs. For fish, assimilation and digestive costs were the same in both coastal and inland populations. These results support the hypothesis that snakes with specialized diets can evolve physiological traits to extract nutrients more efficiently. However, there was no apparent trade-off on the more generalized diet that was associated with this specialization.

Metabolic and blood gas dependence on digestive state in the Savannah monitor lizard Varanus exanthematicus: an assessment of the alkaline tide.

A large alkaline tide (up to 20 mmol l(-1) increase in bicarbonate concentration [HCO3-] with an accompanied increase in blood pH) has previously been reported for some carnivorous reptiles within 24 h after ingesting a large meal. This phenomenon has been attributed to the secretion of large amounts of H+ ions into the stomach, which is required for digestion of large prey items. To test the generality of this phenomenon in carnivorous reptiles, this study quantified the metabolic and acid-base status of the Savannah monitor lizard, Varanus exanthematicus, during digestion at 35 degrees C. Following a meal of approximately 10% of body mass, V(O2) and V(CO2) were measured continuously and arterial pH, blood gases and strong ions were measured every 8 h for 5 days. During peak digestion (24 h post feeding), V(O2) and V(CO2) increased to approximately threefold fasting values (V(O2), 0.95-2.57 ml min(-1) kg(-1); V(CO2) 0.53-1.63 ml min(-1) kg(-1)) while respiratory exchange ratio (R) remained constant (0.62-0.73). During digestion, arterial P(CO2) increased (from 4.6 kPa to 5.8 kPa), and [HCO3-] also increased (from 24.1 mmol l(-1) to 40.3 mmol l(-1)). In contrast to early studies on crocodilians, arterial pH in V. exanthematicus remained relatively stable during digestion (7.43-7.56). Strong ions contributed little to the acid-base compensation during the alkalosis. Collectively the data indicate that the metabolic alkalosis associated with H+ secretion (as indicated by increased plasma bicarbonate) is partially compensated by a respiratory acidosis.