Ribonuclease T1 cleaves RNA after guanosines within single-stranded gaps of any length.

RNA-oligonucleotides with defined single-stranded stretches were designed to investigate the minimal requirements of a ribonuclease T1 substrate. It could be shown, that RNase T1 cleaves single-stranded RNA after a unique guanosine flanked by two double-stranded areas. However, the turnover of such a G-gap is significantly lower than that of a gap of two, three or four nucleotides.

Analysis of the RNase T1 mediated cleavage of an immobilized gapped heteroduplex via fluorescence correlation spectroscopy.

We report a new method for studying the activity of hydrolytic enzymes. Fluorescence correlation spectroscopy was used to observe online the hydrolyzation of a rhodamine B-labeled substrate by ribonuclease T1. A gapped heteroduplex substrate - a hybrid of a ribooligonucleotide and two smaller complementary deoxyribooligonucleotides - was immobilized via biotin to a streptavidin-coated surface of a coverslip. The reported method opens the possibility to study the cleavage of small substrates differing only slightly in molecular weight from the enzyme reaction product. The use of fluorescence correlation spectroscopy allows the detection of very low enzyme concentrations (down to 10(-21) mol 0.05 fM of RNase T1, corresponding to about 600 RNase T1 molecules in 0.02 ml).

Phage display of RNase A and an improved method for purification of phages displaying RNases.

Functional ribonuclease A was presented on the surface of the filamentous phage M13 by fusion to the minor coat protein. RNase activity of the fusion protein was shown by a zymogram assay. In addition, we established a modified method for preparing RNase-displaying phages without contaminating host RNases.

Comorbidity of diabetes and eating disorders. Does diabetes control reflect disturbed eating behavior?

OBJECTIVE: This multicenter study was designed to explore the prevalence of clinical and subclinical eating disorders (EDs), the extent of intentional omission of insulin and oral antidiabetic agents, and its relationship to glycemic control in an inpatient and outpatient population of men and women with type 1 and type 2 diabetes. RESEARCH DESIGN AND METHODS: Data have been collected from 12 diabetes medical centers in two German cities. In a questionnaire and interview-based study, a sample of male and female patients (n = 341 type 1, n = 322 type 2) was assessed for the following eating disorders: anorexia nervosa, bulimia nervosa, binge eating disorder, and eating disorder not otherwise specified. For lack of interview data of several patients meeting the screening criteria, prevalence ranges were calculated. RESULTS: The overall prevalence range of current EDs was 5.9-8.0% (lifetime prevalence 10.3-14.0%). When patients were stratified according to type 1 and type 2 diabetes, there was no difference in prevalence of EDs. However, the distribution of the EDs was different in both types of diabetes, with a predominance of binge eating disorder in the type 2 diabetes sample. Type 1 (5.9%) and type 2 (2.2%) diabetic patients reported deliberate omission of hyperglycemic drugs (insulin or oral agents) in order to lose weight. Compared with control subjects, neither the presence of EDs nor insulin omission influenced diabetic control. CONCLUSIONS: There seems to be no difference in prevalence rates of EDs in both types of diabetes; however, distribution of EDs is different. The findings suggest that neither EDs nor insulin omission are necessarily associated with poor control of glycemia. Binge eating disorder seems to precede type 2 diabetes in most patients and could be one of the causes of obesity that often precedes type 2 diabetes.

Retroperitoneal bronchogenic cyst presenting as an adrenal mass.

Subdiaphragmatic bronchogenic cysts are rare, and those located retroperitoneally are exceptional. A review of the English-language literature revealed only three reported cases. We describe an additional case of a retroperitoneal bronchogenic cyst that presented uniquely as a symptomatic adrenal mass and discuss the cases of subdiaphragmatic bronchogenic cysts reported in the English-language literature.

Cloning and nucleotide sequencing of genes for small, acid-soluble spore proteins of Bacillus cereus, Bacillus stearothermophilus, and "Thermoactinomyces thalpophilus".

As found previously with other Bacillus species, spores of B. stearothermophilus and "Thermoactinomyces thalpophilus" contained significant levels of small, acid-soluble spore proteins (SASP) which were rapidly degraded during spore germination and which reacted with antibodies raised against B. megaterium SASP. Genes coding for a B. stearothermophilus and a "T. thalpophilus" SASP as well as for two B. cereus SASP were cloned, their nucleotide sequences were determined, and the amino acid sequences of the SASP coded for were compared. Strikingly, all of the amino acid residues previously found to be conserved in this group of SASP both within and between two other Bacillus species (B. megaterium and B. subtilis) were also conserved in the SASP coded for by the B. cereus genes as well as those coded for by the genes from the more distantly related organisms B. stearothermophilus and "T. thalpophilus." This finding strongly suggests that there is significant selective pressure to conserve SASP primary sequence and thus that these proteins serve some function other than simply amino acid storage.

Activation and germination characteristics observed in endospores of thermophilic strains of Bacillus.

The properties of endospores of some thermophilic strains of Bacillus were examined. Included were strains isolated from thermal pools and springs in Yellowstone National Park, a strain of B. thermodenitrificans and two strains of B. stearothermophilus, ATCC 7953 (smooth) and T-10. The spores of thermophilic strains of Bacillus contained relatively high levels of dipicolinic acid ranging from 11-14.8% of the spore dry weight, while the calcium levels were similar to those observed in other bacterial endospores including mesophilic bacilli and thermophilic actinomycetes. Spore populations of thermophilic bacilli could not be effectively germinated in solutions of sodium phosphate alone but germinated well in solutions supplemented with one of a variety of organic compounds. Solutions containing L-valine or L-leucine were particularly effective. A wide range of pH permitted the germination of fractions of spore populations, however, optimum germination was observed only at pH values of 6.0 and above. A range in incubation temperatures of less than 25 degrees C permitted 50% or more of the spores of each of the organisms to germinate. Freshly prepared spore did not germinate, but these spores germinated rapidly and completely if they were heated for 30 min at 100 degrees C just prior to germination testing, i.e., the spores were heat activatable. However, spores of thermophilic bacilli could also be activated by shifting them to and holding them at temperatures below their optimum growth temperature of 65 degrees C. Of the ten temperatures tested, ranging from 4 degrees C through 50 degrees C, the optimum reduced temperature for spore activation was 30 degrees C.

[Vulnerability of early postnatal differention processes of the retina and the teratogenic effect of cyclophosphamide (Endoxan)]. / Vulnerabilität früher postnataler Differenzierungsvorgänge der Retina und teratogener Effekt der Zyklophosphamids (Endoxan)

During the postnatal period, cyclophosphamide produces in the retina of rats and mice rosettes containing malformed photoreceptor cells arranged in half-circle formations. The rosettes form only after injections given during the first 4 days post partum and only in the receptor layer which contains desmosomes. After this period, the retina becomes resistant to rosette formation. Cytological changes occur in the form of intense nuclear invaginations and membrane whorls, which may be regarded as compensatory processes.

Glutamic acid decarboxylase in spores of Bacillus megaterium and its possible involvement in spore germination.

Spores of Bacillus megaterium were examined for glutamic acid decarboxylase (GAD). Although dormant spores showed no GAD activity, spores given sonic treatment and heat-activated spores had high activities when assayed for this enzyme. Several parameters of GAD in heat-activated spores were examined. The effects of KCN, NaN(3), 2,4-dinitrophenol, and KF on GAD activity were examined. Only KCN was an effective inhibitor of GAD activity in heated spores and was also shown to be the only effective inhibitor of GAD activity in vegetative bacteria. Similar patterns of inhibition were obtained with GAD activity and with spore germination, KCN being the only effective inhibitor of both, although at different concentrations. Spore GAD activity in heat-activated spores showed a loss with storage at 4 C; on the other hand, storage at 25 C was not accompanied by a loss, but, to the contrary, showed an increase in GAD activity of about 30%. A comparison of GAD activity at different times during germination, growth, and sporulation showed it to be highest in freshly germinated spores. Although vegetative cells contained GAD activity, the level in log-phase cells was approximately one-half the level obtained with freshly germinated spores. Heat-activated mutant spores with a requirement of gamma-aminobutyric acid for germination gave no GAD activity. GAD activity appeared in mutant spores after germination and increased to levels comparable to parent spores after 9 min of germination.