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Liquid-tissue behavior and differential cohesiveness during chick limb budding.

Emerging chick limb-buds at first grow only in length, not width. The growth parameters of limb mesoderm--cell shapes, distributions, division patterns and cleavage orientations--are incompatible with representations of this tissue as an elongating solid composed of proliferating but immobile cells. We observe that samples of both early limb mesoderm and also surrounding flank mesoderm round up like liquid droplets in organ culture. Therefore, liquid-like tissue rearrangments, including cell shuffling movements and neighbor exchanges, may occur in limb and flank mesoderm during in vivo limb budding. If so, differences in limb-flank surface tension properties would have to be present to keep these two fluid cell populations segregated into distinct tissues and properly positioned underneath limb and flank ectoderm. Previous studies have shown that tissue surface tensions are reflected in the spreading behavior of fused pairs of cell aggregates. To determine whether or not they possess differing surface tension properties, we pair excised pieces of early leg-bud, wing-bud or intervening flank mesoderm with pieces of 5 3/4-day heart or liver in hanging drop cultures. For more rapid determinations of relative liquid-tissue cohesiveness than can be obtained in conventional, long-term experiments, aggregate pairs are fixed shortly after fusion. Since partial-envelopment configurations depend upon relative aggregate sizes as well as their tissue surface tensions, new procedures are used to deduce relative aggregate cohesiveness from cross-sections of these briefly fused aggregate pairs. The envelopment tendencies of aggregates fixed 6--9 h after fusion are similar to those fixed 15--19 h after fusion: heart tends to surround leg; heart and wing surround each other with similar frequencies, but flank tends to surround heart. Also, liver tends to surround leg and wing, but flank tends to surround liver. When the effects of relative aggregate size are taken into account, these non-random, tissue-specific patterns of aggregate envelopment indicate that the relative cohesiveness of these tissues falls into the sequence: leg greater than heart congruent to wing greater than liver greater than flank. The in vitro behavior of early limb-bud and neighboring flank mesoderm in these studies suggests that they are not simply mechanically identical portions of a single liquid tissue. We have previously proposed that early limb-bud mesoderm may act like a non-dispersing, cohesive liquid droplet which is embedded within a less cohesive fluid layer of flank tissue (and which is molded distally into paddle-shaped conformations by solid-like limb ectoderm and/or subjacent extracellular matrix). This proposal is not only compatible with the growth parameters of limb-bud mesoderm in vivo, but is also consistent with our observation that flank mesoderm surrounds tissues which surround limb mesoderm in these aggregate-fusion experiments. Our model suggests that differences in the surface tension properties of limb vs...

Animals

A Simple Method to Analyze Context- and Tissue-Specific Cis-Regulatory Modulations of Homeotic (HOX) Genes Using ChIP.

Homeobox genes (HOX), the master regulators, deploy a unique set of target genes to coordinate and orchestrate the spatiotemporal development of an organism. HOX encoded transcriptional factors regulate the expression of target genes by binding to the specific sequences on the genome. Chromatin Immunoprecipitation (ChIP) and Chromatin Immunoprecipitation with Sequencing (ChIP-Seq) are widely used to map and understand specific gene locus and global regulatory regions on the genome. ChIP is a powerful technique of cross-linking the proteins bound to the DNA, fragmenting DNA to the desired size, and pulling them down using specific antibodies to enrich and analyze the protein-bound DNA. Based on the mapping information, a differential ChIP can be performed to understand cis-regulatory modulations at a defined locus by two developmental stages. This chapter describes the differential ChIP used to identify new targets by comparing two different developmental stages simultaneously using Drosophila melanogaster.

Animals

Free amino acids in tissues of the skate Raja erinacea and the stingray Dasyatis sabina: effects of environmental dilution.

Concentrations of individual free amino acids were determined in various tissues of the skate (Raja erinacea) and the stingray (Dasyatis sabina), and the relationship of cellular free amino acid concentrations to intracellular osmoregulation was investigated by adapting these elasmobranchs to half-strength seawater. Each tissue characteristically had high levels of certain specific amino acids. Skate sing muscle contained high concentrations of sarcosine and beta-alanine, skate heart had high concentrations of taurine, and skate erythrocytes had high levels of taurine and beta-alanine. Amino acid levels in skate plasma were very low. High concentrations of aturine and glutamate were found in stingray brain. Adaptation of skates and stingrays to half-strength seawater was accompanied by significant decreases in concentrations of the major free amino acids in skate wing muscle and erythrocytes and in stingray brain, but not in skate heart. The data suggest that in these elasmobranchs certain specific free amino acids are selectively involved in intracellular osmoregulatory mechanisms.

Adaptation, Physiological

A search for blood protozoans in the American woodcock.

Smears were prepared from heart blood of 55 woodcock collected in central Maine between spring and fall, 1972 and 1973. Peripheral blood taken from the wings of 41 of these birds also was examined. Examination of stained films revealed no infected blood cells. Samples of heart blood from 35 of the 41 woodcock were injected into young ducks, quail, and a gull. Plasmodia were not seen in inoculated birds.

Animals

Sensitivity of small subcutaneous vessels to altered respiratory gases and local pH.

Television microscopy was used to quantitate the responses of small arteries and veins, in the wings of unanesthetized bats, to alterations in the inspired concentrations of O2 and CO2. Mean arterial pressure, heart rate, and the diameters of small arteries (28-54 mum) and veins (50-128 mum) were measured during a 90-min protocol--30 min with an inspiratory gas mixture of 20% O2 and 80% N2 (control period); 30 min with a gas mixture containing 5% O2 (hypoxic period) or 12, 20, or 28% CO2 (hypercapnic period); and 30 min with the original control gas. The hypoxic responses were dilatation of arteries and no change in the veins in both innervated and surgically denervated wings. Hypercapnia resulted in artery dilatation in innervated wings. Hypercapnia resulted in artery dilatation in innervated wings and constriction in denervated wings. The veins constricted in both innervated and denervated wings during the hypercapnia period. In another series, topical application of Krebs solutions (pH ranging from 7.7 to 6.7) to exposed segments of small arteries and veins produced dilatation of both vessels with decreasing pH. Artery dilatation during hypoxia and vein constriction during hypercapnia involve non-neural mechanisms, while both a neural stimulus for dilatation and a non-neural stimulus for constriction are components in the response of innervated arteries to hypercapnia. The non-neural stimulus for artery and vein constriction during hypercapnia is not a local decrease in pH.

Animals

Mechanism of cardiovascular effects of clonidine in conscious and anesthetized rabbits.

The actions of intravenous clonidine [2-(2,6 dichlorophenylamino)-2-imidazoline hydrochloride] on blood pressure and heart rate were examined in conscious rabbits. Complete transection of thecervical spinal cord increased the intensity and duration of the hypertensive effect of 30 microgram/kg of clonidine and completely abolished the fall in blood pressure. Heart rate slowing by clonidine was reduced. Result were similar 1 hour, 24 hours and 7 days after cord transection. Bilateral aortic sinus nerve section (baroreceptor deafferentation) increased both the hypertensive and hypotensive action of clonidine but reduced the bradycardia. When cervical cord transection was combined with batensive action, were abolished. We conclude that whereas the hypertensive action results from a direct effect on peripheral adrenoceptors, the fall in blood pressure is related to a reduction in sympathetic tone mediated at the level of the brain stem or more rostrally. The heart rate slowing results from both a reduction in sympathetic tone and also an enhanced vagal outflow. The increase in vagal tone seems to be dependent on the intergrity of baroreceptor afferent pathways.

Animals

Turnover of muscle protein in the fowl. Collagen content and turnover in cardiac and skeletal muscles of the adult fowl and the changes during stretch-induced growth.

The collagen content and the rate of collagen synthesis were measured in the anterior and posterior latissimus dorsi muscles and in heart from fully grown fowl. This was done by measuring the proline/hydroxyproline ratios in the muscle and by a constant infusion of [(14)C]proline. These measurements were also made during the hypertrophy of the anterior muscle in response to the attachment of a weight to one wing of the fowl. In the non-growing muscles the collagen content was higher in the anterior muscle (22.8% of total protein) than in the posterior muscle (9.5% of total protein) and lowest in the heart (3.8% of total protein). In the two skeletal muscles a little over half of the collagen was accounted for by internal collagen (i.e. perimysium and endomysium). Collagen synthesis in these non-growing muscles occurred at 0.59%/day in each of the two skeletal muscles and at 0.88%/day in the cardiac muscle. During hypertrophy the collagen content of the anterior muscle increased, but not as fast as intracellular protein, so that after 58 days the concentration had fallen from 22.8 to 14.4% of total protein. This may have resulted from an incomplete production of the epimysial sheath, since the concentration of internal collagen did not fall and as a result accounted for over 80% of the total in the enlarged muscle. Collagen synthesis increased 8-fold during the first week of the hypertrophy, but never amounted to more than 4% of the total muscle protein synthesis. When the net accumulation of collagen is compared with the increased rate of synthesis it is concluded that between 30 and 70% of the newly synthesized collagen may have been degraded.

Animals

Antihypertensive effects of clonidine in tetraplegic subjects devoid of central sympathetic control.

1. The effects of 300 microgram of oral clonidine on blood pressure, heart rate, plasma noradrenaline and adrenaline concentrations were studied in seven tetraplegic subjects with physiologically complete cervical spinal cord transections. 2. Clonidine did not significantly change resting blood pressure during the 8 h of the study. Resting heart rate fell. Resting plasma noradrenaline and adrenaline concentrations, when measured 2 h after clonidine, were not significantly lower. 3. Urinary bladder stimulation resulted in a marked rise in blood pressure accompanied by an elevation in plasma noradrenaline but not adrenaline. The hypertensive response to bladder stimulation was substantially reduced by clonidine, the maximum suppression occurring 2-4 h after administration of the drug. The plasma noradrenaline response to bladder stimulation, when measured 2 h after clonidine, was significantly lower than the response before clonidine. In two tetraplegic subjects with indwelling catheters the rise in intravesical pressure caused by bladder stimulation was unchanged by clonidine. 4. The pressor response to intravenous noradrenaline in two tetraplegic subjects and to intravenous phenylephrine in a further two tetraplegic subjects was unchanged by clonidine. 5. Clonidine may have additional antihypertensive effects which occur independently of actions in the brain. These effects may be exerted by an action either on sympathetic neurones in the spinal cord or on presynaptic alpha-adrenoreceptors in the periphery.

Adult

Innervation of heart and alary muscles in Sphinx ligustri L. (Lepidoptera). A scanning and transmission electron microscopic study.

The origin and orientation of the heart nerves in Sphinx ligustri and Ephestia kuehniella were investigated by scanning electron microscopy using a special technique which involved pinning the dissected specimens on a stabilizing metal pad. The heart and alary muscles in Sphinx particularly their caudal extremity were also examined by transmission electron microscopy. The alary muscles form an incomplete sheath around the heart with a mainly longitudinal fibre orientation, e.i. antagonistically to the fibres of the heart itself. The heart and alary muscles are multiterminally innervated by branches of the transverse segmental nerves. All branches contain a single electron lucent axon; the thickest branches also possess several neurosecretory axons. Swellings of the segmental nerves may indicate the position of nerve cell bodies. There are no lateral heart nerves. Only one type of neuromuscular junction is abundant in the alary muscles but less frequently found in the heart. The terminals originate from the central axon only. They are capped by glial cells, which interdigitate with the muscle cells. They penetrate into the T-system toward the Z-discs and form a complex intercellular space system. Exocytosis of dense-cored vesicles into this "perisynaptic reticulum" seems likely. Sites of neurohaemal release are distributed along the nerve branches and special nerve endings occur at the level of the ostia. The possible nervous influence upon heart activity is discussed.

Animals

Early work load tests for evaluation of long-term prognosis of acute myocardial infarction.

Exercise tests performed 3 and 9 weeks after acute myocardial infarction in 205 patients were found to give prognostic information on the survival during a follow-up period of 2 to 5 years. The appearance of tachycardia, major ventricular arrhythmias, or anginal complaints during these early exercise tests was thus accompanied by a significantly increased mortality during the observation period. Ventricular arrhythmias disclosed by exercise proved to be of higher prognostic significance than those recorded at rest on the same occasions. The usefulness of early exercise tests in the evaluation of the response to antiarrhythmic treatment after acute myocardial infarction as well as of the prognostic importance of the effects was documented in a smaller series of patients.

Acute Disease

The central hypotensive effect of clonidine. Studies in tetraplegic subjects.

A single oral dose of 300 microng of clonidine lowered systolic blood pressure by 20 +/- 4 mm Hg and diastolic blood pressure by 13 +/- 4 mm Hg in five healthy normotensive subjects (controls). Heart rate fell from 56 +/- 2 to 52 +/- 2 beats/min. In six tetraplegic subjects with physiologically complete chronic cervical spinal cord transection above the level of the sympathetic outflow, the same dose of clonidine did not significantly lower either systolic or diastolic blood pressure. Heart rate fell from 67 +/- 4 to 53 +/- 2 beats/min. Peak plasma concentrations of clonidine, measured by mass fragmentography, and elimination of the drug from plasma were similar in tetraplegic and control subjects and there was no difference in the incidence of the principal side effects of clonidine--sedation and dry mouth. Although the number of subjects studied is small, the absence of a fall in blood pressure after clonidine in the tetraplegic subjects suggests that the hypotensive action of clonidine in man is dependent on intact descending bulbospinal pathways and is mediated by withdrawal of sympathetic tone and provides direct evidence that some antihypertensive drugs may lower blood pressure in man by a direct action on the brain.

Adult

Pseudomonas maltophilia causing heroin-associated infective endocarditis.

The association of Pseudomonas maltophilia endocarditis in three patients with recent history of intravenous drug abuse is reported. All three patients had abnormal heart valves (two prosthetic and one rheumatic). A prominent characteristic of this uncommon pathogen is its in vitro resistance to the commonly used antimicrobials. Cure was achieved in all three cases. In two cases, synergistic antibiotic combinations were used. In one case, plasmid-mediated resistance to amikacin sulfate (Amikan, British; no comparable US product) emerged during therapy. The two patients with prosthetic valves received combined surgical and antibiotic therapy.

Adult

Clonidine withdrawal in hypertension. Changes in blood-pressure and plasma and urinary noradrenaline.

Treatment was interrupted abruptly in 6 hypertensive patients receiving clonidine 0-45-5-4 mg daily. Blood-pressure rose to pretreatment levels within 24-48 h of withdrawal and was accompanied by insomnia, headache, flushing, sweating, and apprehension. These symptoms began 18-20 h after the last dose of clonidine. Plasma-noradrenaline levels and urinary catecholamine excretion increased 24-72 h after withdrawal of clonidine. The subjective symptoms were most prominent in patients on higher doses (greater than 1 mg/day) and in those who had previously been receiving treatment with other antihypertensive drugs. One patient on a very low daily dose (0-15 mg) of clonidine had no symptoms and no significant changes in blood-pressure or catecholamine production after drug withdrawal.

Adult

Physical activity as an index of heart attack risk in college alumni.

Risk of first heart attack was found to be related inversely to energy expenditure reported by 16,936 Harvard male alumni, aged 35-74 years, of whom 572 experienced heart attacks in 117,680 person-years of followup. Stairs climbed, blocks walked, strenuous sports played, and a composite physical activity index all opposed risk. Men with index below 2000 kilocalories per week were at 64% higher risk than classmates with higher index. Adult exercise was independent of other influences on heart attack risk, and peak exertion as strenuous sports play enhanced the effect of total energy expenditure. Notably, alumni physical activity supplanted student athleticism assessed in college 16-50 years earlier. If it is postulated that varsity athlete status implies selective cardiovascular fitness, such selection alone is insufficient to explain lower heart attack risk in later adult years. Ex-varsity athletes retained lower risk only if they maintained a high physical activity index as alumni.

Adult

Pharmacokinetic and concentration-effect relationships of clonidine in essential hypertension.

The effect of oral doses of 300 microgram of clonidine hydrochloride on blood pressure, sedation and saliva production in 5 essential hypertensives were qualitatively similar to the effects in normotensive subjects. Peak plasma clonidine concentration (1.34 +/- 0.28 ng/ml) and plasma half-life (10.0 +/- 0.8 h) were similar to normotensives. During chronic oral dosing there was no evidence of drug accumulation. Some tolerance to the sedative and salivary flow effects occurred but no tolerance to the hypotensive effect was observed. There was a linear relationship between reduction in saliva flow and plasma levels of clonidine. The hypotensive effect was also related to plasma level at low concentrations. At plasma levels greater than 1.5 ng/ml the hypotensive effect was diminished. This loss of effect at high plasma concentration may be related to the peripheral, post-synaptic alpha-adrenoceptor agonist action of the drug.

Blood Pressure

Cell movement and the mechanism of invasiveness: a survey of the behaviour of some normal and malignant cells implanted into the developing chick wing bud.

A survey of the behaviour of a variety of normal and malignant tumours and cells has been carried out to gain insights into the mechanisms of tumour invasiveness. The tumours and cells were implanted into the developing chick wing bud, which is a loose mesenchyme bounded by ectoderm. The distribution of the grafted cells was examined histologically after one or two days. The special feature of this assay is that the behaviour of cells is tested in a 3-dimensional tissue. Cells from 3 different carcinomas, mouse lung tumour, rat bladder tumour and human breast tumour did not invade the mesenchyme, whereas trophoblast, sarcoma 180, cultured hamster fibroblasts (BHK, PyBHK, Nil 8, HSV Nil 8) and neuroblastoma cells did. Cells from embryonic pigmented retina and heart ventricle were non-invasive. These results suggest that cell movement may not be a common feature of all invasive tumours. The cells that did move into the mesenchyme appeared to do so by various mechanisms. Lack of contact inhibition of movement, although probably involved in the invasiveness of sarcoma 180 cells, does not appear to be necessary for invasion: cells that have been shown to exhibit contact inhibition of movement (BHK and PyBHK) also invade. Both normal and transformed cells (BHK and PyBHK; Nil 8 and HSV Nil 8) moved into the mesenchyme. Other invading cells, such as trophoblast, neuroblastoma and to a small extent, HSV Nil 8 cells, destroy the adjacent host tissue and this may be important in the invasiveness of these cells. The patterns of invasion and interactions with the host tissue were varied. Trophoblast and the fibroblasts were often elongated along the basement membrane at the ectoderm/mesenchyme border and also closely apposed to the endothelial linings of blood vessels. Sarcoma 180 and neuroblastoma cells clustered around nerves. The embryonic tissues and neuroblastoma cells were often associated with blood vessels. These results are discussed in relation to tumour invasion. A striking finding was that the carcinoma cells were frequently found positioned within the wing ectoderm on the basement membrane. This affinity of carcinoma cells for the epithelium rather than the mesenchyme leads to a reappraisal of the mechanisms involved in the invasiveness of carcinomas.

Animals