Gene cloning, nucleotide sequence and biochemical properties of a cytoplasmic cyclomaltodextrinase (neopullulanase) from Alicyclobacillus acidocaldarius, reclassification of a group of enzymes.

A gene encoding a cyclomaltodextrinase (neopullulanase) was cloned from the thermoacidophilic bacterium Alicyclobacillus acidocaldarius ATCC27009 and its nucleotide sequence was determined. The encoded CdaA protein lacked an N-terminal signal sequence and aligned well with a family of bacterial proteins described as maltogenic alpha-amylases, neopullulanases or cyclomaltodextrinases. Escherichia coli cells harboring the cloned cdaA gene produced a 66-kDa protein that degraded pullulan in a sodium dodecyl sulfate-polyacrylamide gel. A. acidocaldarius cells grown on maltose, soluble starch or pullulan synthesized the same protein. Neopullulanase activity of the protein was cytoplasmic and its pH optimum of 5.5 was close to the pH value of the cytoplasm. CdaA degraded cyclomaltodextrins rapidly and pullulan (to panose) more slowly. It is proposed that CdaA functions as a cytoplasmic cyclomaltodextrinase (EC 3.2.1.54).

Acidostable and acidophilic proteins: the example of the alpha-amylase from Alicyclobacillus acidocaldarius.

Acidophilic microorganisms grow optimally at pH values between 1-4. They have adapted to the acid condition by maintaining their cytoplasmic pH at a value close to neutrality. Hence, only those (macro)-molecules, which face the acid medium, have had to adapt to this extreme condition. Literature data show that several exoproteins from thermoacidophilic prokaryotes are characterized by a low charge density. It is proposed that this property contributes to the stability of these proteins both below and above the pKa-values of their glutamate and aspartate residues. As an example of an acidophilic protein, the alpha-amylase from the Gram-positive Alicyclobacillus acidocaldarius ATCC27009 was studied. The enzyme is thermoacidophilic, with optima of temperature and pH of 75 degrees C and pH 3, respectively. The nucleotide sequence of the cloned gene (8) indicates that the alpha-amylase belongs to a large family of starch-degrading enzymes with a characteristic catalytic (beta alpha)8-domain. Three essential and probably catalytic acidic residues have been conserved, suggesting that the acidophilic alpha-amylase degrades starch with essentially the same mechanism as do its neutrophilic relatives. Still, the acidophilic protein contains three exchanges in residues uniformally or almost uniformally conserved among all members of the enzyme family. In order to test whether these exchanges contribute to the acidic pH optimum, the alpha-amylase gene was expressed in Escherichia coli. Sonication of the enzyme-producing cells released alpha-amylase activity associated with a 140 kDa protein. The optima of temperature and pH for the protein produced in E. coli were similar to those of the native enzyme. Experiments are underway in which it is tested which residues contribute to the acid pH optimum of the alpha-amylase.

Activation-dependent expression of low affinity IgG receptors Fc gamma RII(CD32) and Fc gamma RIII(CD16) in subpopulations of human T lymphocytes.

Receptors for IgG (Fc gamma R) are expressed by small subpopulations of peripheral blood T lymphocytes. Our studies demonstrate that T lymphocytes can be induced in vitro to express two different low-affinity Fc gamma R. Mitogen activation of peripheral blood T lymphocytes obtained from eight healthy individuals leads to considerable augmentation of the Fc gamma RIII+ (CD32) T cell subpopulation. The highest percentage of CD32 expressing T lymphocytes could be detected after three days of activation. The T cell subpopulation which transiently express the CD32 antigen, encompasses CD4+ and CD8+ cells. Molecular cloning of the CD32 antigen by reverse transcription and polymerase chain reaction demonstrates that activated human T lymphocytes express the Fc gamma RIIIb2 isoform. The percentage of the Fc gamma RIII+ (CD16) T cell subpopulation was significantly increased only in the lymphocyte populations obtained from three out of eight volunteers immediately after mitogen activation. However, during short-term cell culture the CD16 expressing CD8+ T cell subset increased in the T cell population from all individuals investigated. During this time, the IL-2 receptor alpha-chain (CD25) expression level decreased as a function of time. In contrast to the CD8+CD16+ T cells, the percentage of the non-MCH-restricted CD56+CD16+ T cells was not influenced by mitogen activation and time of cell cultivation. We could show that CD16 in T cells is able to mediate a stimulus leading to proliferation of the CD8+CD56-CD16+ T cells but not that of the CD56+CD16+ T cell subset. This discrepancy cannot be explained by the expression of different Fc gamma RIII isoforms, because both T cell subsets express Fc gamma RIIIA alpha, as we demonstrate in this report.

Erythrocyte sodium transport in normotensive and hypertensive patients on chronic hemodialysis, acute effect of treatment.

Patients on chronic hemodialysis were divided into two groups: normotensive patients (group I) and renal hypertensive patients treated with antihypertensives (group II). The sodium and potassium contents in red blood cells ([Na+]i, [K+]i), ouabain-resistant net sodium uptake (ORNa+ uptake, phi Na), the relative ORNa+ uptake (k), the mean cell hemoglobin concentration (MCHC), and acid-base status were examined just before and after dialysis. The results indicate that in treated renal hypertensive patients k is stimulated and causes lower red blood cell sodium content. The reason for this increase remains obscure: the pattern of alterations of the sodium transport components during dialysis is similar in all patients: [Na+]i and phi Na increase significantly during dialysis, and the increases in [Na+]i are closely related to increases in pH and bicarbonate.

[Saralasin test in the differential diagnosis of essential and renal hypertension]. / Der Saralasintest in der Differentialdiagnostik essentieller und renaler Hypertonien.

In a multicentric prospective study should be tested clinically the effectiveness and the tolerance of an angiotensin-II-antagonist (Saralasin-IWF) developed by the Institut für Wirkstofforschung der Akademie der Wissenschaften, its position in the differential-diagnostic step programme of the arterial hypertension should be analysed and with it should be performed pathogenetic investigations for hypertension after kidney transplantation. Taking into consideration international studies our results confirm that the Saralasin test, taking into account strongly standardized methodical prerequisites, is suited to objectify a participation of the RAAS in the hypertension pathogenesis, without, however, thus making an absolutely reliable evidence concerning the etiology of hypertension. The Saralasin test may represent an important diagnostic criterion for an optimization of the therapy of "volume-resistant" hypertension under the conditions of haemodialysis and in connection with selective renin determinations it possesses a high value in the screening diagnostics of the arterial stenosis after allogenic kidney transplantation.

[Studies and results of the use of nuclear medicine methods in kidney function tests]. / Untersuchungen und Ergebnisse beim Einsatz nuklearmedizinischer Methoden zur Nierenfunktionsdiagnostik.

It is sought for alternative methods for the endogenous creatinine clearance as parameters for the GFR. For this purpose served investigations with Tc-99m-Sn-DTPA. It was found a good reproducibility for the slope clearance with 3 blood takings between 60 and 120 minutes. When combined with the camera function scintigraphy a side-separated quantitative statement of the clearance equivalent is possible. The inulin clearance serves as reference method. The investigations at the gamma-camera with Tc-99m-Sn-DTPA also allow the determination of the perfusion which is particularly of interest in patients with hypertension. According to the comparison at the gamma camera a diagnostic schema is recommended.