Distribution of viral abundance in the reef environment of Key Largo, Florida.

The distribution of viral and microbial abundance in the Key Largo, Fla., reef environment was measured. Viral abundance was measured by transmission electron microscope direct counts and plaque titer on specific bacterial hosts in water and sediment samples from Florida Bay (Blackwater Sound) and along a transect from Key Largo to the outer edge of the reef tract in Key Largo Sanctuary. Water column viral direct counts were highest in Blackwater Sound of Florida Bay (1.2 x 10(7) viruses per ml), decreased to the shelf break (1.7 x 10(6) viruses per ml), and were inversely correlated with salinity (r = -0.97). Viral direct counts in sediment samples ranged from 1.35 x 10(8) to 5.3 x 10(8)/cm(3) of sediment and averaged nearly 2 orders of magnitude greater than counts in the water column. Viral direct counts (both sediment and water column measurements) exceeded plaque titers on marine bacterial hosts (Vibrio natriegens and others) by 7 to 8 orders of magnitude. Water column viral abundance did not correlate with bacterial direct counts or chlorophyll a measurements, and sediment viral parameters did not correlate with water column microbial, viral, or salinity data. Coliphage, which are indicators of fecal pollution, were detected in two water column samples and most sediment samples, yet their concentrations were relatively low (<2 to 15/liter for water column samples, and <2 to 108/cm(3) of sediment). Our findings indicate that viruses are abundant in the Key Largo environment, particularly on the Florida Bay side of Key Largo, and that processes governing their distribution in the water column (i.e., salinity and freshwater input) are independent of those governing their distribution in the sediment environment.     

Modulation of SPARC Expression during Butyrate-Induced Terminal Differentiation of Cultured Human Keratinocytes: Regulation via a TGF-β-Dependent Pathway

Expression of SPARC (secreted protein acidic and rich in cysteine), a 43-kDa extracellular matrix-associated glycoprotein involved in tissue remodeling, was quantitated during normal human keratinocyte (NHK) growth in culture and as a function of sodium n-butyrate (NaB)-induced differentiation to mature enucleate cornified envelopes (CEs). Low levels of SPARC expression were observed in the basal-like cells of control NHKs, with isolated cells showing intense SPARC expression on the ventral surface. After addition of NaB, SPARC expression increased and the pattern of expression shifted to one involving predominantly suprabasal cells (i.e., spinous cells, pre-CEs, and mature CEs). Dense deposits of SPARC often surrounded the mature CEs. Flow cytometric analysis indicated that approximately 13% of NHKs expressed SPARC within 24 h of seeding into culture. This fraction of SPARC⁺ cells increased with time and peaked immediately postconfluence (31.3 ± 6.3% SPARC⁺). Cellular SPARC expression then decreased to baseline levels during entrance into plateau phase growth. SPARC was detectable in all phases of the cell cycle. SPARC levels were more intense and heterogeneous within the G₂/M and G₁ phases while S phase cells exhibited relatively homogeneous, low intensity, SPARC expression. During NaB-induced NHK differentiation, SPARC intracellular content increased prior to the onset of CE formation (i.e., 2 days after its addition) followed by a period of extracellular accumulation which coincided with the time of maximal CE generation (i.e., Days 4 and 5 after NaB addition). Correlation of cell size with anti-SPARC immunoreactivity revealed a predominance of SPARC expression in cells with a suprabasal phenotype. NHKs cultured on fibronectin (FN), an established modulator of epidermal cell maturation in vitro, showed a similar response to NaB. In general, however, the level of NaB-induced SPARC expression was considerably reduced in FN cultures correlating with a lower efficiency of CE formation. Induced SPARC expression was, in large part, dependent on autocrine transforming growth factor-β (TGF-β) production since incubation in the presence of NaB + neutralizing antibodies to TGF-β inhibited both the expression of SPARC by 72% and development of mature CEs.     

Direct Cloning of Yeast Genes from an Ordered Set of Lambda Clones in Saccharomyces Cerevisiae by Recombination in Vivo

We describe a technique that facilitates the isolation of yeast genes that are difficult to clone. This technique utilizes a plasmid vector that rescues lambda clones as yeast centromere plasmids. The source of these lambda clones is a set of clones whose location in the yeast genome has been determined by L. Riles et al. in 1993. The Esherichia coli-yeast shuttle plasmid carries URA3, ARS4 and CEN6, and contains DNA fragments from the lambda vector that flank the cloned yeast insert. When yeast is cotransformed with linearized plasmid and lambda clone DNA, Ura(+) transformants are obtained by a recombination event between the lambda clone and the plasmid vector that generates an autonomously replicating plasmid containing the cloned yeast DNA sequences. Genes whose genetic map positions are known can easily be identified and recovered in this plasmid by testing only those lambda clones that map to the relevant region of the yeast genome for their ability to complement the mutant phenotype. This technique facilitates the isolation of yeast genes that resist cloning either because (1) they are underrepresented in yeast genomic libraries amplified in E. coli, (2) they provide phenotypes that are too marginal to allow selection of the gene by genetic complementation or (3) they provide phenotypes that are laborious to score. We demonstrate the utility of this technique by isolating three genes, GAL83, SSN2 and MAK7, each of which presents one of these problems for cloning.     

Phospholipase A2 inhibitors from marine algae

Twelve out of twenty-nine compounds isolated from benthic marine algae from the phyla Chlorophyta, Phaeophyta and Rhodophyta have been found to be potent inhibitors of bee venom derived phospholipase A₂ (PLA₂) (> 50%) in the M range. The compounds investigated were from: Bryopsis pennata, Rhipocephalus phoenix, Caulerpa prolifera, C. racemosa, C. bikinensis, Cymopolia barbata, Laurencia cf. palisada, Laurencia sp., Ochtodes crockeri, Liagora farinosa, Sphaerococcus coronipifolius, Phacelocarpus labillardieri, Dictyota sp., B furcaria galapagensis, Stypopodium zonale, Dictyopteris undulata, Stoechospermum marginatum, Dictyopteris divaricata, Dilophus fasciola and Dilophus sp. This is the first report of bee venom PLA₂ inhibition in vitro by pure compounds isolated from marine algae.     

A new technique to reduce internal phosphorus loading by in-lake phosphate fixation in shallow lakes

A new, flexible, fast, robust and economic technique was developed to treat sediment in shallow lakes with phosphate binding chemicals. The upper 0.15 m of the sediment is thoroughly mixed with ferric chloride using a water-jet manifold coupled to a dosing pump and a navigation control system. Its logistics were tried out in a small, shallow and hypertrophic peat lake, Lake Groot Vogelenzang.     

Cell wall synthesis during growth and maturation of Nitella internodal cells

An improved ¹³C-density-labeling method was used to study cell wall synthesis in rapidly expanding, slowly expanding and recently mature internodes of Nitella translucens var axillaris (A.Br.) R.D.W. As cells matured, the rate of wall synthesis slowed and the deposition of cellulose microfibrils changed from a predominantly transverse direction in the primary wall of rapidly expanding internodes to a helicoidal array in the secondary wall of mature internodes. The secondary wall was characterized by relatively higher rates of cellulose synthesis and lower rates of pectin synthesis than the primary wall. The synthesis of xyloglucan also decreased markedly at the transition to secondary wall synthesis, while the synthesis of mannose-rich hemicellulose increased. Even though structural differences were striking between the primary and secondary walls of Nitella, compositional differences between the two types of wall were quantitative rather than qualitative. The authors appreciate the assistance of Martin Yousef with the electron microscopy.     

Experimental candidiasis in Japanese quail: Pathological changes

Candidiasis was experimentally produced in young Japanese quail by oral administration ofCandida albicans cells. Lesions were confined to upper digestive tract with most characteristic changes occurring on the mucosa of crop. No lesions were observed in other tissues of the body. The initial changes in the crop were characterized by thickening and yellowish-white necrotic plaques on the mucosa. From 10th day onwards, there was marked thickening and corrugations of the crop mucosa giving it a typical turkish towel appearance. Varying degree of mucosal swelling was also observed in the oesophagus and proventriculus. Two of the infected birds also revealed yellowish-white necrotic plaques on the tongue at 7th and 10th day post-infection. The prominent microscopic lesions in the crop and tongue consisted of hyperkeratosis and parakeratosis with congestion of the subepithelial tissues. Varying degree of parakeratosis and epithelial hyperplasia coupled with subepithelial oedema and hypertrophy of glands was observed in the oesophagus. The proventriculus and small intestine revealed congestion, oedema, mild to marked goblet cell hyperplasia and focal epithelial sloughing. Fungal elements could be demonstrated in the sections of tongue upto 10 days while in crop upto 14 days post-infection. Reisolation of the fungus was consistently achieved from the crop of infected birds throughout the duration of the experiment.     

Topography of short portal vessels in the rat pituitary gland: a scanning electron-microscopic and morphometric study of corrosion cast replicas

We applied scanning electron microscopy combined with imaging and morphometric techniques to analyze the dorsal topography and morphology of short portal vessels linking the capillary beds of the pituitary neural and anterior lobes in adult male albino rats. The pituitary microvasculature was replicated by intracarotid injection of Batson's No. 17 compound producing plastic casts that were advantageous for comprehensive morphometric analyses using an imaging device. The analysis revealed the existence of two types of portal vessels having quantitatively different morphological properties. The bilateral venular plexus of 3–4 vessels located at the base of the infundibular stalk (each venule measuring 300 m in length and 32 m in diameter) appears to be the major part of the short portal system in the dorsum of the rat pituitary gland. Narrower capillary-like shunt vessels (6.8 m in diameter), of about the same length as the venules, were situated throughout other subregions of the intermediate lobe cleft. The short portal vessels of both types made direct anastomoses with the capillary networks in the neural and anterior lobes. The neural lobe capillaries were twice as numerous (1324 per mm²), and only half as wide (6.2 m), as the sinusoidal capillaries in the anterior lobe (density of 637 per mm²; diameter of 13.7 m). The topographical position of the portal venular system suggests that the caudolateral subregions of the pituitary neural and anterior lobes have a functional relationship dependent on rapid interlobe transfer of neurohumoral factors such as hormones via the portal blood. This process appears to be supplemented throughout the rest of the cleft between the two lobes by a small number of capillary shunts that supply the epithelial cell lobules of the intermediate lobe in situ. The findings collectively indicate that this portal system provides a constant stream of neurohumoral information that is shared moment-by-moment between the pituitary neural and anterior lobes.     

Cholinesterases regulate neurite growth of chick nerve cells in vitro by means of a non-enzymatic mechanism

Cholinesterases present homologies with some cell adhesion molecules; however, it is unclear whether and how they perform adhesive functions. Here, we provide the first direct evidence showing that neurite growth in vitro from various neuronal tissues of the chick embryo can be modified by some, but not all, anticholinesterase agents. By quantifying the neuritic G4 antigen in tectal cell cultures, the effect of anticholinesterases on neurite growth is directly compared with their cholinesterase inhibitory action. BW 284C51 and ethopropazine, inhibiting acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), respectively, strongly decrease neurite growth in a dose-dependent manner. However, echothiophate which inhibits both cholinesterases, does not change neuritic growth. These quantitative data are supplemented by morphological observations in retinal explant cultures grown on striped laminin carpets, viz., defasciculation of neurite bundles by BW 284C51 and Bambuterol occurs, indicating that these drugs disturb adhesive mechanisms. These data strongly suggest that a) cholinesterases can participate in regulating axonal growth, b) both AChE and BChE can perform such a nonsynaptic function, and c) this function is not the result of the enzyme activity per se, since at least one drug was found that inhibits all cholinesterase activities but not neurite growth. Thus, a secondary site on cholinesterase molecules must be responsible for adhesive functions.