Characteristics of extracellular proteases produced by Bacillus laterosporus and Flavobacterium sp. isolated from gelatinfactory effluents

Forty bacterial isolates from the effluents of a gelatin factory (Jabalpur, India) were screened for protease activity and the two most potent producers were identified as Bacillus laterosporus and a Flavobacterium sp. The enzymes of both isolates were optimal at pH 8 and 60°C, with maximum activity after 90 min. The enzyme activity of B. laterosporus was suppressed by Fe²⁺, Mg²⁺, Mn²⁺ and Zn²⁺ ions but was enhanced by Ba²⁺ and Ca²⁺. That of Flavobacterium sp. was suppressed by Mg²⁺ and Mn²⁺ ions but enhanced by Ba²⁺, Ca²⁺ and Fe²⁺. The enzyme activity of the former was strongly inhibited by KCN, whereas that of the latter was only slightly inhibited by 8-hydroxyquinoline.     

Use of pregnancy-specific protein-B and estrone sulfate for determination of pregnancy on Day 49 in fallow deer (Dama dama )

The objective of this study was to determine if pregnancy specific protein-B (PSPB) and estrone sulfate (E(1)SO(4)) could be used to determine pregnancy status in fallow deer (Dama dama ). Forty mature does were synchronized for estrus with an intravaginal progesterone-releasing device (CIDR) and then artificially inseminated via laparoscopy with frozen semen on one day. Ultrasound examination and jugular blood sampling were done 49 days later. Transrectal ultrasonography was done to presumptively determine the pregnancy status at the time of blood sampling. Serum estrone sulfate concentrations were significantly (P < 0.05) greater in pregnant (n=31) than nonpregnant (n=9) females at 49 days of gestation (166.7 +/- 25.9 pg/ml vs 36.3 +/- 11.1 pg/ml, respectively). The percentage of [(125)I]PSPB bound was significantly (P < 0.01) lower when sera of pregnant (n=29) versus nonpregnant (n=9) females was added to RIA tubes (63.7 +/- 1.6% vs 98.1 +/- 1.6%, respectively). There were 30 fawns born from the group of females that were diagnosed pregnant based on ultrasound. We conclude that estrone sulfate and PSPB can be used to determine pregnancy status in fallow deer at 49 days of gestation.     

Alterations in the U3 region of the long terminal repeat of an infectious thymotropic type B retrovirus.

We isolated and characterized a type B thymotropic retrovirus (DMBA-LV) which is highly related to mouse mammary tumor virus (MMTV) isolates and which induces T-cell thymomas with a high incidence and a very short latent period. Regions of nonhomology between the DMBA-LV genome and the MMTV genome were identified by heteroduplex mapping and nucleotide sequence studies. In the electron microscope heteroduplex mapping studies the EcoRI-generated 5' and 3' fragments of the DMBA-LV genome were compared with the corresponding fragments of the MMTV (C3H and GR) genome isolated from mammary tumors. The results indicated that DMBA-LV contained a region of nonhomologous nucleotide sequences in the 3' half of the U3 region of the long terminal repeat (LTR). Nucleotide sequence studies confirmed these results and showed that in this region 440 nucleotides of the MMTV (C3H) sequences were deleted and substituted with a segment of 122 nucleotides. This substituted segment in the form of a tandem repeat structure contained nucleotide sequences derived exclusively from sequences which flanked the substitution loop. The distal glucocorticoid regulatory element was unaltered, and two additional copies of the distal glucocorticoid regulatory element-binding site were present in the substituted region. The restriction endonuclease map of the reconstructed molecular clone of DMBA-LV was identical to that corresponding to unintegrated linear DMBA-LV DNA present in DMBA-LV-induced tumor cell lines. Since the nucleotide sequences of the LTRs present in four different DMBA-LV proviral copies isolated from a single thymoma were identical, we concluded that they were derived from the same parental virus and that this type B retrovirus containing an alteration in the U3 region of its LTR could induce thymic lymphomas. Thus, DMBA-LV represents the first example of a productively replicating type B retrovirus that contains an LTR modified in the U3 region and that has target cell and disease specificity for T cells.     

Cloning and characterization of DPPL1 and DPPL2, representatives of a novel type of mammalian phosphatidate phosphatase

Phosphatidate phosphatase (PAP) enzymes are classified as either Mg(2+)-dependent (PAP1) or Mg(2+)-independent (PAP2) with respect to their Mg(2+) cofactor requirement for catalytic activity. Sensitivity to the thioreactive compound N-ethylmaleimide (NEM) has also been used to differentiate PAP1 (NEM-sensitive) from PAP2 (NEM-insensitive) activity in mammalian cells. We report here the cloning and initial characterization of DPPL1 and DPPL2, representatives of a novel type of mammalian phosphatidate phosphatase. Both DPPL1 and DPPL2 show greater homology to a yeast diacylglycerol pyrophosphate (DGPP) phosphatase, DPP1, than to known phosphatidate phosphatases of mammals. Like the yeast DPP1 protein, both DPPL1 and DPPL2 proteins show broad substrate specificity, but DGPP is the preferred substrate compared with LPA and PA. These reactions are Mg(2+)-independent, but unlike DPP1 and mammalian PAP2, they are sensitive to NEM. DPPL1 mRNA is ubiquitously expressed in various tissues and cells, but DPPL2 mRNA is restricted to several tissues including the brain, kidney and testis, and it is preferentially expressed in endothelial cells. Immunohistological staining of synovium containing vessels, plasma cells and lymphocytes revealed specific expression of DPPL2 protein in the endothelium. Collectively, our work indicates that DPPL1 and DPPL2 represent a novel type of mammalian phosphatidate phosphatase.     

Needle in a haystack: microdissecting the proteome of a tissue

Summary. Laser-assisted microdissection is a recent technology that enables cells to be harvested from tissue sections. Proteins can be extracted from the dissected cells for molecular analysis. This enables the analysis of proteins in specific cell types in an in vivo system. Although quantities of protein obtained from the dissected material can be small, it is possible to use established methods such as Western Blotting and 2D-PAGE, as well as newer technologies such as SELDI-MS, to analyse the proteins. This review describes the applications and technical considerations for using laser-assisted dissected cells in proteomics research.     

In vivo specific tension of human skeletal muscle.

In this study, we estimated the specific tensions of soleus (Sol) and tibialis anterior (TA) muscles in six men. Joint moments were measured during maximum voluntary contraction (MVC) and during electrical stimulation. Moment arm lengths and muscle volumes were measured using magnetic resonance imaging, and pennation angles and fascicular lengths were measured using ultrasonography. Tendon and muscle forces were modeled. Two approaches were followed to estimate specific tension. First, muscle moments during electrical stimulation and moment arm lengths, fascicular lengths, and pennation angles during MVC were used (data set A). Then, MVC moments, moment arm lengths at rest, and cadaveric fascicular lengths and pennation angles were used (data set B). The use of data set B yielded the unrealistic specific tension estimates of 104 kN/m(2) in Sol and 658 kN/m(2) in TA. The use of data set A, however, yielded values of 150 and 155 kN/m(2) in Sol and TA, respectively, which agree with in vitro results from fiber type I-predominant muscles. In fact, both Sol and TA are such muscles. Our study demonstrates the feasibility of accurate in vivo estimates of human muscle intrinsic strength.     

Properties of the ATP phosphohydrolase of the facultative thermophile Bacillus coagulans

Abstract The thermostability of the ATP phosphohydrolase of the facultative thermophile Bacillus coagulans has been investigated. Fractionation of disintegrated cell suspensions by differential centrifugation revealed a similar distribution of enzyme activity irrespective of growth temperature. Most of the activity was located in the membrane fraction. Thermostability of solubilized (BF₁) preparation from cells grown at 37°C or 55°C was similar, but membrane-bound BF₀BF₁ from 37°C-grown cells was inactivated at lower temperatures than that from 55°C-grown cells.
Inhibition of the membrane-bound (BF₀BF₁)ATPase by 4-chloro-7-nitro-benzofuran (NbfCl) and quercetin, which both act on the BF₁ portion of the enzyme, was different from that seen with the soluble (BF₁) enzyme. The results show that some modification of BF₁ must occur when the enzyme is membrane-bound.