Machine milking of dairy goats during lactation: udder anatomy, milking characteristics, and blood concentrations of oxytocin and prolactin

Forty-four goats were milked and milk flow recorded without or with 1 min manual prestimulation in early, mid and late lactation. Ultrasound measurements of cross sections of the whole mammary gland were performed in a water bath. In additional experiments with 15 goats, milk flow was recorded and frequent blood samples were taken for the determination of oxytocin and prolactin concentrations. Milk yield increased from the first to the third lactation and decreased markedly during the course of lactation. Average and peak milk flow rates were closely related to the actual milk yield. The ultrasound cisternal area was 27.4 +/- 1.5% of the entire udder half cross section. Milking characteristics were scarcely different without or with prestimulation, although oxytocin was released within 30 s after the start of prestimulation, whereas oxytocin concentrations without prestimulation increased only after the start of milking. Concentrations of prolactin were higher during July and August than in April, and increased similarly with or without prestimulation during milking. In contrast to dairy cows, prestimulation and an opportune release of oxytocin during milking does not significantly influence the course of milk flow in goats, and this is probably because large amounts of cisternal milk allow milk ejection to be induced only after the start of milking without causing bimodal or otherwise reduced milk flow.     

The comparative amino acid sequences, substrate specificities and gene or cDNA nucleotide sequences of some prokaryote and eukaryote amidinotransferases: implications for evolution

The amino acid sequences of the amidinotransferases and the nucleotide sequences of their genes or cDNA from four Streptomyces species (seven genes) and from the kidneys of rat, pig, human and human pancreas were compared. The overall amino acid and nucleotide sequences of the prokaryotes and eukaryotes were very similar and further, three regions were identified that were highly identical. Evidence is presented that there is virtually zero chance that the overall and high identity regions of the amino acid sequence similarities and the overall nucleotide sequence similarities between Streptomyces and mammals represent random match. Both rat and lamprey amidinotransferases were able to use inosamine phosphate, the amidine group acceptor of Streptomyces. We have concluded that the structure and function of the amidinotransferases and their genes has been highly conserved through evolution from prokaryotes to eukaryotes. The evolution has occurred with: (1) a high degree of retention of nucleotide and amino acid sequences; (2) a high degree of retention of the primitive Streptomyces guanine + cytosine (G + C) third codon position composition in certain high identity regions of the eukaryote cDNA; (3) a decrease in the specificities for the amidine group acceptors; and (4) most of the mutations silent in the regions suggested to code for active sites in the enzymes.     

The xenoestrogen bisphenol A induces growth, differentiation, and c-fos gene expression in the female reproductive tract

The xenoestrogen bisphenol A (BPA) has been shown to mimic estrogen both in vivo and in vitro. BPA stimulates PRL secretion and the expression of a PRL regulating factor from the posterior pituitary in the estrogen-sensitive Fischer 344 rat (F344), but not in Sprague-Dawley (SD) rats. The goal of the present studies was to examine the in vivo actions of BPA on the reproductive tract. The specific objectives were 1) to characterize the short term effects of BPA on cell proliferation and c-fos expression in the uterus and vagina, and 2) to compare the effects of prolonged exposure to low doses of BPA on the reproductive tract of F344 and SD rats. Treatment with single high doses of BPA induced cell proliferation in the uterus and vagina of ovariectomized F344 rats, as determined by bromodeoxyuridine immunostaining. This proliferation was dose dependent (from 37.5-150 mg/kg) and followed a time course similar to that of estradiol (E2). Quantitative RT-PCR revealed that both BPA and E2 increased c-fos messenger RNA levels in the uterus 14- to 16-fold within 2 h, which returned to basal levels after 6 h. In the vagina, BPA-induced c-fos expression remained elevated for up to 6 h, compared with the transient increase caused by E2. Treatment of F344 rats for 3 days with continuous release capsules that supplied a much lower dose of BPA (approximately 0.3 mg/kg x day) resulted in hypertrophy, hyperplasia, and mucus secretion in the uterus and hyperplasia and cornification of the vaginal epithelium. The reproductive tract of SD rats did not respond to this treatment paradigm with BPA. These studies demonstrate that 1) the molecular and morphological alterations induced by BPA in the uterus and vagina are nearly identical to those induced by estradiol; 2) the vagina appears to be especially sensitive to the estrogenic actions of BPA; 3) the reproductive tract of the inbred F344 rat appears more sensitive to BPA than that of the outbred SD rat; and 4) continuous exposure to microgram levels of BPA is sufficient for exerting estrogenic actions.     

Monocyte procoagulant activity induced by adherence to an artificial surface is reduced by end-point immobilized heparin-coating of the surface

Heparin-coating improves the biocompatibility of blood contacting artificial surfaces. This led us to investigate the impact of heparin-coating (Carmeda AB, Stockholm) of polymetylmetacrylate on the expression of monocyte tissue factor procoagulant activity (TF-PCA) by surface adhesion. Also, the anticoagulant effect of heparin-coating in the presence or absence of adherent procoagulant monocytes was assessed. This is of particular interest, since activation of extrinsic coagulation by adherent monocyte TF-PCA may play a significant role in thrombin generation during extracorporeal circulation. Monocytes exposed to heparin-coated or non-coated polymetylmetacrylate expressed TF-PCA. The heparin coat did not affect the rate of monocyte adhesion. However, heparin-coating reduced the induction of TF-PCA of non-adherent and adherent monocytes by 17 and 33% (p <0.001 and p <0.0003), respectively. Heparin-coating in the absence of monocytes, totally inhibited the clotting of recalcified plasma (p <0.003). In contrast, in the presence of adherent monocytes expressing TF-PCA, surface-bound heparin did not inhibit clotting. However, inclusion of heparin in a plasma concentration of 8.9 IU/ml totally inhibited the activation of coagulation. It is apparent that heparin-coating of an artificial surface is an efficient means to inhibit coagulation of recalcified plasma, but much less so when procoagulant monocytes are adherent to the coated surface. The present findings are of clinical relevance, since monocytes will adhere to blood contacting surfaces of extracorporeal circuits or to implanted vascular prostheses and subsequently express TF-PCA, and this may promote thromboembolism.     

Protein fluctuations are sensed by stimulated infrared echoes of the vibrations of carbon monoxide and azide probes

The correlation functions of the fluctuations of vibrational frequencies of azide ions and carbon monoxide in proteins are determined directly from stimulated photon echoes generated with femtosecond infrared pulses. The asymmetric stretching vibration of azide bound to carbonic anhydrase II exhibits a pronounced evolution of its vibrational frequency distribution on the time scale of a few picoseconds, which is attributed to modifications of the ligand structure through interactions with the nearby Thr-199. When azide is bound in hemoglobin, a more complex evolution of the protein structure is required to interchange the different ligand configurations, as evidenced by the much slower relaxation of the frequency distribution in this case. The time evolution of the distribution of frequencies of carbon monoxide bound in hemoglobin occurs on the approximately 10-ps time scale and is very nonexponential. The correlation functions of the frequency fluctuations determine the evolution of the protein structure local to the probe and the extent to which the probe can navigate those parts of the energy landscape where the structural configurations are able to modify the local potential energy function of the probe.     

Three cases of tuberculosis after heart transplantation in Spain

The fibrinolytic capacity of patients with acute myocardial infarction (AMI) is known to be impaired. The primary regulatory element of the fibrinolytic system is plasminogen activator inhibitor (PAI). It has been previously observed that there are 2 peaks in the plasma PAI level of AMI patients at 4h and 16h after thrombolytic therapy with recombinant tissue plasminogen activator (rtPA). Lanoteplase/SUN9216 is a mutant tPA with a biological half-life longer than that of rtPA. Thrombolytic therapy with mutant tPA or rtPA was carried out consecutively in 21 patients with AMI (8 patients as the mutant tPA group, and 13 patients as the rtPA group). The recanalization time of the mutant tPA group was significantly faster than that of the rtPA group (16.1 +/- 3.9 min vs 39.6 +/- 4.8 min, p<0.01). The PAI activity at 4h after the initiation of thrombolysis was significantly lower in the mutant tPA group than in the rtPA group (8.74 +/- 5.46IU/L vs 26.74 +/- 3.35 IU/L, p<0.01). There was a one mild peak in serial plasma PAI activity levels 24h after the initiation of thrombolysis. The results suggest that thrombolytic therapy with mutant tPA reduced the impairment of fibrinolytic capacity. The mutant tPA gives faster recanalization and lower PAI activity after successful thrombolysis, compared with rtPA.     

Adenosine A1 receptor stimulation antagonizes the negative inotropic effects of the PKC activator dioctanoylglycerol

It has been suggested that adenosine cardioprotection occurs via adenosine A1 receptor-mediated activation of protein kinase C (PKC). However, adenosine has well-known vasodilatory effects in the myocardium, whereas PKC is a vasoconstrictor. This study examined whether adenosine A1 receptor activation alters the effects of the PKC activator. 1,2-dioctanoyl-s,n-glycerol (DOG) in isolated perfused rat hearts (left-ventricular developed pressure) and rat ventricular myocytes ([Ca2+]i and cell shortening). Exposure to DOG decreased left-ventricular developed pressure by 30%, an effect that was completely reversible. Pretreatment of isolated hearts with either the PKC inhibitor chelerythrine or the adenosine A1 agonist 2-chloro-N6-cyclo-cyclo-isolated pentlyadenosine (CCPA) attenuated the negative inotropic effects of DOG. In the isolated myocytes, DOG decreased [Ca2+]i and cell shortening by 25 and 28%, respectively, effects that were attenuated by both chelerythrine and CCPA. The CCPA attenuation of the DOG-induced decrease in [Ca2+]i and cell shortening was blocked by pretreating the myocytes with the adenosine A1 antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). These results indicate that in rat ventricular myocardium, adenosine A1 receptor activation attenuates the apparent PKC-dependent negative inotropic effects of DOG via preservation of [Ca2+]i levels.     

Efficient directional cloning of recombinant adenovirus vectors using DNA-protein complex

We describe an efficient cloning system utilizing adenoviral DNA-protein complexes which allows the directional cloning of genes into adenoviral expression vectors in a single step. DNA-protein complexes derived from a recombinant adenovirus (AVC2.null) were isolated by sequential use of CsCl step gradients followed by isopycnic centrifugation in a mixture of CsCl and guanidine HCl. AVC2.null is an adenoviral expression vector containing unique restriction sites between the human CMV-IE promoter and the SV40 intron/polyadenylation site. Transgenes were prepared for cloning into this vector by introduction of compatible restriction sites by PCR. A vector expressing rat granulocyte-macrophage colony-stimulating factor (GM-CSF) was constructed using DNA-protein complex as well as by traditional recombination techniques. The efficacy of our adenoviral cloning system utilizing DNA-protein complex was two logs higher than that seen using homologous recombination. All viruses generated by directional ligation of the insert into the vector DNA-protein complexes contained the desired transgene in the correct orientation. This technique greatly simplifies and accelerates the generation of recombinant adenoviral vectors.     

Cytosolic and nuclear distribution of PPARgamma2 in differentiating 3T3-L1 preadipocytes

In light of the pivotal role that PPARgamma2 plays in the expression of fat specific genes (e.g., A-FABP), we have examined the hypothesis that a rise in PPARgamma2 protein is required for the expression of A-FABP, and that the acceleration of fat cell differentiation by the thiazolidinedione agent, pioglitazone (PIOG), reflects an increase in the abundance of PPARgamma2 mRNA and protein. Western analyses surprisingly revealed that undifferentiated 3T3-L1 fibroblasts contained significant levels of PPARgamma2 protein; that the amount of total cellular PPARgamma2 only increased 2-fold during differentiation; and that the levels of PPARgamma2 protein and mRNA were not increased by PIOG even though fat cell differentiation was accelerated by PIOG as revealed by a 20-fold increase in A-FABP expression. Cell fractionation studies revealed that PPARgamma2 was evenly distributed between the cytosolic and nuclear compartments in both undifferentiated and differentiating 3T3-L1 cells. Immunocytochemical studies with a PPARgamma2-specific antibody indicated that PPARgamma2 was diffusely distributed throughout the cytosol of undifferentiated 3T3-L1 cells, but as the differentiation progressed, the PPARgamma2 became focused around the developing lipid droplets. In contrast to PPARgamma2, undifferentiated 3T3-L1 cells contained no measurable quantities of RXRalpha, but once fat cell differentiation was initiated by treatment with IBMX and dexamethasone, the cellular content of RXRalpha increased several fold. The rise in RXRalpha content paralleled the induction of A-FABP, but the expression of RXRalpha was not enhanced by PIOG. Although the amount of PPARgamma2 and RXRalpha was unaffected by PIOG, gel shift assays revealed that PIOG stimulated PPARgamma2/RXRalpha binding to the adipose response element of A-FABP by 5-fold in less than 12 h. Apparently, RXRalpha rather than PPARgamma2 is the pivotal trans-factor essential for the initiation of terminal fat cell differentiation. However, the high cytsolic content of PPARgamma2 and its association with the lipid droplet of differentiating 3T3-L1 cells suggests PPARgamma2 may possess a cytosolic function in the developing fat cell.     

The synthesis of symmetrical and unsymmetrical P1/P1' cyclic ureas as HIV protease inhibitors

Cyclic urea SD146, a potent HIV protease inhibitor bearing a flat resistance profile, possessed poor solubility and bioavailability, which precluded further development of the compound. In an effort to improve upon the pharmacokinetic profile of the compound, several analogs modified at the P1/P1' residues were prepared and evaluated. Several of those compounds displayed significant improvement of physical properties.