Genomic analysis of the mouse protamine 1, protamine 2, and transition protein 2 gene cluster reveals hypermethylation in expressing cells

To understand the role of chromatin structure in the expression of the mouse protamine 1, protamine 2, and transition protein 2 genes during spermatogenesis, we have examined the genomic organization of this cluster of ``haploid-specific' genes. As seen in the human genome, protamine 2, transition protein 2, and approximately 2.8 kb of a CpG island, hereafter called CpG island-dTP2, were clustered in a small region. Methylation analyses of this region have demonstrated that i) unlike most other tissue-specific genes, the protamine 1, protamine 2, and transition protein 2 genes were located in a large methylated domain in round spermatids, the cell type where they are transcribed, ii) the protamine 1 gene was only partially methylated in somatic cells and in testes from 7-day-old mice, and iii) the approximately 2 kb upstream and downstream of the CpG island-dTP2 were only partially methylated in somatic tissues. DNase I analysis revealed the presence of at least five strong DNase I hypersensitive sites over the CpG island-dTP2 in somatic tissues, but not in germ cells, and sequence analysis indicated that the CpG island-dTP2 is homologous to a CpG island located approximately 10.6 kb downstream of the human transition protein 2 gene. Although the nature of a CpG island-dTP2 and the function of a CpG island-dTP2-containing somatic tissue-specific DNase I hypersensitive sites in close proximity to the germ cell-specific gene cluster are unclear, the ``open' chromatin structure of the CpG island-dTP2 may be responsible for the partial methylation pattern of the flanking sequences including the transition protein 2 gene in somatic tissues. Received: 6 September 1996 / Accepted: 14 January 1997     

Evaluation of mean skin temperature formulas by infrared thermography

 To study the reliabiliity of formulas for calculating mean skin temperature (T _sk), values were computed by 18 different techniques and were compared with the mean of 10,841 skin temperatures measured by infrared thermography. One hundred whole-body infrared thermograms were scanned in ten resting males while changing the air temperature from 40° C to 4° C. Local, regional average and mean skin temperatures were obtained using an image processing system. The agreement frequency, defined as the percentage of the calculated T _sk values which agreed with the corresponding infrared thermographic T _sk within ±0.2° C, ranged for with the various formulas from 7% to 80%. In many sites, the local skin temperature did not coincide with the regional average skin temperature. When the local skin temperatures which showed the highest percentage similarity to the regional average skin temperature within ±0.4° C were applied to the formula, the agreement frequency was markedly improved for all formulas. However, the agreement frequency was not affected by changing the weighting factors from specific constants to individually measured values of regional surface area. By applying the physiologically reliable accuracy range of ±0.2° C in the moderate and ±0.4° C in the cool condition, agreement frequencies of at least 95% were observed in formulas involving seven or more skin temperature measurement sites, including the hand and foot. We conclude that calculation of a reliable mean skin temperature must involve more than seven skin temperature measurement sites regardless of ambient temperature. Optimal sites for skin temperature measurement are proposed for various formulas. Received: 2 December 1996 / Accepted: 25 June 1997     

Mutational analysis of the three cysteines and active-site aspartic acid 103 of ketosteroid isomerase from Pseudomonas putida biotype B.

In order to clarify the roles of three cysteines in ketosteroid isomerase (KSI) from Pseudomonas putida biotype B, each of the cysteine residues has been changed to a serine residue (C69S, C81S, and C97S) by site-directed mutagenesis. All cysteine mutations caused only a slight decrease in the k(cat) value, with no significant change of Km for the substrate. Even modification of the sulfhydryl group with 5,5'-dithiobis(2-nitrobenzoic acid) has almost no effect on enzyme activity. These results demonstrate that none of the cysteines in the KSI from P. putida is critical for catalytic activity, contrary to the previous identification of a cysteine in an active-site-directed photoinactivation study of KSI. Based on the three-dimensional structures of KSIs with and without dienolate intermediate analog equilenin, as determined by X-ray crystallography at high resolution, Asp-103 was found to be located within the range of the hydrogen bond to the equilenin. To assess the role of Asp-103 in catalysis, Asp-103 has been replaced with either asparagine (D103N) or alanine (D103A) by site-directed mutagenesis. For D103A mutant KSI there was a significant decrease in the k(cat) value: the k(cat) of the mutant was 85-fold lower than that of the wild-type enzyme; however, for the D103N mutant, which retained some hydrogen bonding capability, there was a minor decrease in the k(cat) value. These findings support the idea that aspartic acid 103 in the active site is an essential catalytic residue involved in catalysis by hydrogen bonding to the dienolate intermediate.     

Methanogenesis in thermophilic biogas reactors

Methanogenesis in thermophilic biogas reactors fed with different wastes is examined. The specific methanogenic activity with acetate or hydrogen as substrate reflected the organic loading of the specific reactor examined. Increasing the loading of thermophilic reactors stabilized the process as indicated by a lower concentration of volatile fatty acids in the effluent from the reactors. The specific methanogenic activity in a thermophilic pilot-plant biogas reactor fed with a mixture of cow and pig manure reflected the stability of the reactor. The numbers of methanogens counted by the most probable number (MPN) technique with acetate or hydrogen as substrate were further found to vary depending on the loading rate and the stability of the reactor. The numbers of methanogens counted with antibody probes in one of the reactor samples was 10 times lower for the hydrogen-utilizing methanogens compared to the counts using the MPN technique, indicating that other non-reacting methanogens were present. Methanogens that reacted with the probe againstMethanobacterium thermoautotrophicum were the most numerous in this reactor. For the acetate-utilizing methanogens, the numbers counted with the antibody probes were more than a factor of 10 higher than the numbers found by MPN. The majority of acetate utilizing methanogens in the reactor wereMethanosarcina spp. single cells, which is a difficult form of the organism to cultivatein vitro. No reactions were observed with antibody probes raised againstMethanothrix soehngenii orMethanothrix CALS-1 in any of the thermophilic biogas reactors examined. Studies using 2-¹⁴C-labeled acetate showed that at high concentrations (more than approx. 1 mM) acetate was metabolized via the aceticlastic pathway, transforming the methyl-group of acetate into methane. When the concentration of acetate was less than approx. 1 mM, most of the acetate was oxidized via a two-step mechanism (syntrophic acetate oxidation) involving one organism oxidizing acetate into hydrogen and carbon dioxide and a hydrogen-utilizing methanogen forming the products of the first microorganism into methane. In thermophilic biogas reactors, acetate oxidizing cultures occupied the niche ofMethanothrix species, aceticlastic methanogens which dominate at low acetate concentrations in mesophilic systems. Normally, thermophilic biogas reactors are operated at temperatures from 52 to 56° C. Experiments using biogas reactors fed with cow manure showed that the same biogas yield found at 55° C could be obtained at 61° C after a long adaptation period. However, propionate degradation was inhibited by increasing the temperature.     

Effects of prostaglandins, dibutyryl cAMP, LHRH, estrogens, progesterone, and potassium on output of prostaglandin F2 alpha, 13, 14-dihydro-15-keto-prostaglandin F2 alpha, hCG, estradiol, and progesterone by placental minces

In order to compare the endocrine response of placental minces to luteinizing hormone releasing hormone (LHRH) and dibutyryl cAMP (dbcAMP) and to screen for effects of potential stimulatory and inhibitory substances, the simultaneous outputs of PGF2 alpha, 13, 14-dihydro-15-keto-prostaglandin F2 alpha (PGFM), progesterone, 17 beta-estradiol, and hCG were evaluated during a 4 hour incubation in 5 placentas. The output of hCG was highest for 12-week placentas, intermediate for a 16 week placenta, and lowest for term placentas. The output of 17 beta-estradiol by 12 and 16 week placentas in the presence of 30 microM dehydroepian-drosterone sulfate (DHEAS) was greater than that by term placentas. Progesterone output was apparently independent of gestational age although some variation between 12-week placentas was demonstrated. Output of PGF2 alpha was lower in 12 and 16-week placentas than in term placentas and that of PGFM was lower in 12-week placentas than in term placentas. LHRH (100 nM) produced stimulation of PGF2 alpha output (P less than .005) and a trend toward inhibition of progesterone output (which failed to achieve statistical significance) but no stimulation of hCG under these conditions. Stimulation of the outputs of hCG (P less than .005) and PGF2 alpha (P less than .001) and inhibition of that of progesterone (P less than .005) was produced by 20 mM dbcAMP. DHEAS inhibited output of progesterone (P less than .01) and PGF2 alpha (P less than .01). There were no effects of potassium, estrogens, progesterone, or prostaglandins on output of any measured substance.     

Laboratory Diagnosis of Canine Pyometra

The principal laboratory test used to confirm the pyometra diagnosis in the bitch has been the determination of the total white blood cell count in venous blood. A marked elevation is known to be a characteristic of the disease. In the present study, the white blood cell count was determined as well as the γ-globulin level. An elevation of the γ-globulin level and the total white blood cell count was very characteristic to the pyometra patients. The increase in the number of white blood cells nor the high γ-globulin level cannot be regarded specific for pyometra, therefore it was regarded important to find out a more specific test for pyometra. When sonicated E. coli bacteria were tested against sera from pyometra patients in electroimmunodiffusion, the precipitation was almost always detected when E. coli had been isolated from the uterus. This technique provides a quick method in detecting the causative E. coli infection. The present study suggests that whenever laboratory tests are used to confirm the pyometra diagnosis by the total white blood cell count, it is advantageous to analyze the total γ-globulin level in the serum as well as specific antibodies against a common E. coli antigen. Because of the reliability of the glutaraldehyde coagulation test and the simple technique, this can be suggested as the method of choice for an average small animal practice.     

The production of 6-aminopenicillanic acid by a multistage tubular reactor packed with immobilized penicillin amidase

A theoretical model equation was derived to find the correlation between the conversion and the amount of immobilized penicillin amidase in column. The theoretical values of the conversion were predicted form this correlation and compared with experimental results. It was observed in a column reactor that the pH drop along the column path was linear versus the enzyme loading and that the enzyme activity was also linearly dependent on pH up to 8.0. In order to diminish the effect of pH drop, a continuous two-stage plug-flow reactor (PFR) with pH adjustment between the two columns was used was used in the experiments, and two- and three-stage PFRs were simulated by computer. In the case of the two-stage PFR, the maximum productivity was demonstrated experimentally and theoretically as well. when an equal amount of the immobilized enzyme was packed in both columns. It was also predicted in the tree-stage PFR system that the optimal distributions of enzyme loading in three columns were found to be 1:1:1. It was demonstrated that the increased number of reactors in series could enhance the level of the maximum productivity with a given amount of enzyme loading.