Axial filament formation in Bacillus subtilis: induction of nucleoids of increasing length after addition of chloramphenicol to exponential-phase cultures approaching stationary phase.

When chloramphenicol was added to a culture of Bacillus subtilis in early exponential growth, microscopic observation of cells stained by 4',6-diamidino-2-phenylindole showed nucleoids that had changed in appearance from irregular spheres and dumbbells to large, brightly stained spheres and ovals. In contrast, the addition of chloramphenicol to cultures in mid- and late exponential growth showed cells with elongated nucleoids whose frequency and length increased as the culture approached stationary phase. The kinetics of nucleoid elongation after the addition of chloramphenicol to exponential-phase cultures was complex. Immediately after treatment, the rate of nucleoid elongation was very rapid. The nucleoid then elongated steadily for about 4 min, after which the rate of elongation decreased considerably. Nucleoids of cells treated with 6-(p-hydroxyphenylazo)-uracil (an inhibitor of DNA synthesis) exhibited the immediate rapid elongation upon chloramphenicol treatment but not the subsequent changes. These observations suggest that axial filament formation during stationary phase (stage I of sporulation) in the absence of chloramphenicol results from changes in nucleoid structure that are initiated earlier, during exponential growth.     

Biosynthesis of the corrin macrocycle of coenzyme B12 in Pseudomonas denitrificans.

Studies with cell-free protein preparations from a series of recombinant strains of Pseudomonas denitrificans demonstrated that precorrin-3 is converted into a further trimethylated intermediate, named precorrin-3B, along the pathway to coenzyme B12. It was then shown that the part of the pathway from precorrin-3 (called precorrin-3A hereafter) to precorrin-6x involves three intermediates, precorrin-3B, precorrin-4, and precorrin-5. Precorrin-3B was isolated in its native (reduced) as well as its oxidized (factor-IIIB) states, and precorrin-4 was isolated in its oxidized form only (factor-IV). Both factors were in vitro precursors of precorrin-6x. The synthesis of precorrin-6x from precorrin-3A was shown to be catalyzed by four enzymes, CobG, CobJ, CobM, and CobF, intervening in this order. They were purified to homogeneity. CobG, which converts precorrin-3A to precorrin-3B, was found to be an iron-sulfur protein responsible for the oxidation known to occur between precorrin-3A and precorrin-6x, and CobJ, CobM, and CobF are the C-17, C-11, and C-1 methylases, respectively. The acetate fragment is extruded after precorrin-4 formation. This study combined with our recent structural studies on factor-IV (D. Thibaut, L. Debussche, D. Fréchet, F. Herman, M. Vuilhorgne, and F. Blanche, J. Chem. Soc. Chem. Commun. 1993:513-515, 1993) and precorrin-3B (L. Debussche, D. Thibaut, M. Danzer, F. Debu, D. Fréchet, F. Herman, F. Blanche, and M. Vuilhorgne, J. Chem. Soc. Chem. Commun. 1993:1100-1103, 1993) provides a first step-by-step picture of the sequence of the enzymatic reactions leading to the corrin ring in P. denitrificans.     

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.     

Low species turnover of upland Amazonian birds in the absence of physical barriers

Aim

One of the oldest and most powerful ways for ecologists to explain distinct biological communities is to invoke underlying environmental differences. But in hyper-diverse systems, which often display high species richness and low species abundance, these sorts of community comparisons are especially challenging. The classic view for Amazonian birds posits that riverine barriers and habitat specialization determine local and regional community composition. We test the tacit, complementary assumption that similar bird communities should therefore permeate uniform habitat between major rivers, regardless of distance.

Location

Upland (terra firme) rainforests of central Amazonia.

Methods

We conducted intensive whole-community surveys of birds in three pairs of 100-ha plots, separated by 40–60 km. We then used dissimilarity indices, cluster analysis, and ordination to characterize differences among the six avian communities.

Results

In all, we detected 244 forest-dependent birds, with an average of 190 species (78%) per plot. Species turnover was negligible, no unique indicator species were found among plot pairs, and all documented species were already known from a complete inventory at one of the three sites.

Main Conclusions

Our study corroborates the classic biogeographical pattern and suggests that turnover contributes little to regional avian diversity within upland forests. Using a grain size of 100 ha, this implies that upland birds perceive the environment as uniform, at least over distances of ~60 km. Therefore, to maximize both local species richness and population persistence, our findings support the conservation of very large tracts of upland rainforest. Our analyses also revealed that the avifauna at Reserva Ducke, encroached by urban sprawl from the city of Manaus, shows the hallmarks of a disturbed community, with fewer vulnerable insectivores. This defaunation signals that even an enormous preserve (10 × 10 km) in lowland Amazonia is not insulated from anthropogenic degradation within the surrounding landscape.     

ExbB acts as a chaperone-like protein to stabilize TonB in the cytoplasm

The TonB protein is required to transduce energy from the cytoplasmic membrane to outer membrane transport proteins of Gram-negative bacteria. Two accessory proteins, ExbB and ExbD, are required for TonB function and it has been suggested that TonB and ExbBD form a complex in the membrane. In this paper we demonstrate that there are two spatially distinct, functional interactions between ExbBD and TonB. First, there is an interaction between ExbBD and the N-terminal signal-like peptide of TonB, probabiy the formation of a stable complex in the membrane. Second, ExbB interacts with TonB in the cytoplasm. This interaction involves the domain of TonB that is normally periplasmic. Thus, this is a transient interaction which occurs during the synthesis and/or localization of TonB, implying a chaperone-like role for ExbB. The transmembrane topology of ExbB was shown to be consistent with this role.     

A ribonucleoprotein inclusion body in Entamoeba invadens

Summary Cytochemical tests have shown that the chromatoid bodies contain mainly ribonucleic acid and protein. There is no evidence to suggest the presence of any desoxyribonucleic acid, lipid, glycogen, neutral fat, or metaphosphate. The cyclic changes in the chromatoid body have been studied by means of light and electron microscopy and estimations of the total RNA present. The results indicate that the chromatoid bodies arise by a process of aggregation of small groups of 250–300 A units form polycrystalline masses in precystic and early cysts. At the same time there is a steady rise in the amount of RNA present. In the maturing cysts, the crystalline masses fragment into separate particles, but there is no corresponding fall in the amount of RNA. The significance of these results in relation to the previous literature on chromatoid bodies and similarly named cellular inclusions in spermatogenic and plant cells, as well as in the protozoa, is discussed. Attention is drawn to the striking similarity of composition, formation, development and ultimate fate of these various inclusion bodies but as the terminology is still confused it is proposed that the term chromatoid body be retained solely for particulate or lamellar ribonucleo-protein accumulations. Acknowledgements. Most of this study was presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy at the University of Edinburgh. I should like to express my deep appreciation to Prof. M. M. Swann F. R. S. for his interest and helpful guidance in this work. I am grateful to the Melville Trust for Cancer Research who provided the electron microscope.     

Transforming growth factor-beta 1 acts cooperatively with sodium n-butyrate to induce differentiation of normal human keratinocytes.

Growth factors with established biological activity toward cultured normal human epidermal keratinocytes (NHEKs) (e.g., transforming growth factor-beta, TGF-beta; retinoic acid, RA) initiate programmed changes in cellular maturation which differ with regard to the specific differentiation pathway (normal or abnormal) analyzed. Sodium butyrate (NaB) initiates one form of epidermal differentiation leading to enhanced cornified envelope (CE) formation which involves abrogation of the normally inhibitory effect of RA on NHEK maturation. NaB also induces TGF-beta mRNA in the maturing suprabasal compartment, suggesting that TGF-beta may play a role in NaB-initiated NHEK differentiation. Treatment with TGF-beta 1 alone, however, only marginally increased (by twofold) the number of detergent-resistant CEs compared to control NHEKs and did not alter the prevalence of fully mature enucleated CEs. TGF-beta 1 was quite effective in inducing significant levels of CE expression when used simultaneously with suboptimal concentrations of NaB. The cooperative action of suboptimal NaB and TGF-beta 1 generated numbers of CEs which, in fact, exceeded the incidence of mature CEs formed in response to optimal levels of NaB alone. Neutralizing antibodies to TGF-beta, moreover, effectively reduced the incidence of CE formation in cultures treated with optimal NaB concentrations, further implicating endogenous TGF-beta activity in the NaB-initiated NHEK differentiation model. It is suggested, therefore, that within the NaB-induced pathway of NHEK differentiation, TGF-beta can positively modulate expression of the differentiated phenotype but alone is insufficient for generation of mature CEs.