Chronic silastic central venous catheterization for induction, maintenance and support of persistent granulocytopenia in rabbits

In order to investigate new approaches in diagnosis, prevention and treatment of infectious complicating chemotherapy-induced granulocytopenia, we developed and prospectively evaluated a method of chronic central venous catheterization for the induction, maintenance and support of persistent granulocytopenia in rabbits. The method entails a central venous silastic catheter with a subcutaneous tunnel and a heparin lock device for repeated non-traumatic sampling of blood and administration of medications. During the course of 10 months, 226 rabbits were studied. Mean duration of catheter placement was 27 days, 17 of which were spent in granulocytopenia. Two-way flow was sustained throughout the duration of placement in 205 rabbits (91%) and for 5,845 (95%) of a total 6,163 catheter-days. All but two catheters could be flushed throughout the duration of their placement. Postoperative infectious complications related to catheter insertion developed in less than 1% of the rabbits. This method of chronic catheterization safely provides long-term venous access for studies requiring frequent venous access, including the painless induction, maintenance, and support of chronic granulocytopenia in rabbits.     

Conformation-specific precipitation of human α2-macroglobulin by divalent zinc or calf thymus histone H3

Highly purified native α₂-macroglobulin (α₂M), α₂M-trypsin, and α₂M-methylamine were compared in experiments designed to study protein precipitation. Significant turbidity developed within 30 min in solutions containing histone H3 and either α₂M-methylamine or α₂M-trypsin, as determined by absorbance at λ = 550 nm. No turbidity was detected in solutions that contained histone H3 and native α₂M or histone H3 alone. Experiments with radioiodinated histone H3 or radioiodinated proteinase inhibitor confirmed that both the H3 and the α₂M “fast” forms (α₂M-methylamine, α₂M-trypsin) were present in the precipitates generated. As much as 70% of the ¹²⁵I-α₂M-methylamine was recovered in the precipitate after incubation with a 120-fold molar excess of H3 (concentration of α₂M-methylamine, 0.28 μm). The ratio of histone to proteinase inhibitor by weight in the precipitate was approximately two. Under comparable conditions, somewhat less α₂M-trypsin precipitated from solutions containing H3 than did α₂M-methylamine; however, inactivation of the α₂M-trypsin with phenylmethylsulfonyl fluoride prior to incubation increased the level of precipitation significantly. Solutions containing poly-l-lysine (M_r ~ 13,000) instead of histone did not form precipitates with any of the forms of α₂M studied. In a second set of experiments, radioiodinated native α₂M, α₂M-trypsin, and α₂M-methylamine were incubated in solutions containing ZnCl₂, BaCl₂, CdCl₂, CuSO₄, MgCl₂, or NiCl₂ (concentration of divalent cation between 5 μm and 1.0 mm). Native α₂M was soluble in all of these salts. By contrast, α₂M-methylamine and α₂M-trypsin precipitated extensively from solutions containing greater than 100 μm ZnCl₂. Precipitation was greater than 90% complete at 1 mm ZnCl₂. A similar effect was not observed with any of the other divalent cations.     

Prion Protein Paralog Doppel Protein Interacts with Alpha-2-Macroglobulin: A Plausible Mechanism for Doppel-Mediated Neurodegeneration

Doppel protein (Dpl) is a paralog of the cellular form of the prion protein (PrP^C), together sharing common structural and biochemical properties. Unlike PrP^C, which is abundantly expressed throughout the central nervous system (CNS), Dpl protein expression is not detectable in the CNS. Interestingly, its ectopic expression in the brain elicits neurodegeneration in transgenic mice. Here, by combining native isoelectric focusing plus non-denaturing polyacrylamide gel electrophoresis and mass spectrometry analysis, we identified two Dpl binding partners: rat alpha-1-inhibitor-3 (α₁I₃) and, by sequence homology, alpha-2-macroglobulin (α₂M), two known plasma metalloproteinase inhibitors. Biochemical investigations excluded the direct interaction of PrP^C with either α₁I₃ or α₂M. Nevertheless, enzyme-linked immunosorbent assays and surface plasmon resonance experiments revealed a high affinity binding occurring between PrP^C and Dpl. In light of these findings, we suggest a mechanism for Dpl-induced neurodegeneration in mice expressing Dpl ectopically in the brain, linked to a withdrawal of natural inhibitors of metalloproteinase such as α₂M. Interestingly, α₂M has been proven to be a susceptibility factor in Alzheimer''s disease, and as our findings imply, it may also play a relevant role in other neurodegenerative disorders, including prion diseases.     

Efficient succinic acid production from high-sugar-content beverages by Actinobacillus succinogenes

This study presents the production of succinic acid (SA) by Actinobacillus succinogenes using high-sugar-content beverages (HSCBs) as feedstock. The aim of this study was the valorization of a by-product stream from the beverage industry for the production of an important building block chemical, such as SA. Three types of commercial beverages were investigated: fruit juices (pineapple and ace), syrups (almond), and soft drinks (cola and lemon). They contained mainly glucose, fructose, and sucrose at high concentration—between 50 and 1,000 g/L. The batch fermentation tests highlighted that A. succinogenes was able to grow on HSCBs supplemented with yeast extract, but also on the unsupplemented fruit juices. Indeed, the bacteria did not grow on the unsupplemented syrup and soft drinks because of the lack of indispensable nutrients. About 30–40 g/L of SA were obtained, depending on the type of HSCB, with yield ranging between 0.75 and 1.00 g_SA/g_S. The prehydrolysis step improved the fermentation performance: SA production was improved by 6–24%, depending on the HSCB, and sugar conversion was improved of about 30–50%.     

Continuous intravenous infusion and simultaneous pharmacokinetic monitoring in ambulatory rabbits with a programmable micropump and dual subcutaneous Silastic central venous catheters.

We describe a newly developed method of continuous intravenous infusion and simultaneous monitoring of plasma levels of investigational compounds in ambulatory untethered rabbits. Continuous infusion was administered by means of a portable programmable external micropump which permitted adjustable dosing. Simultaneous plasma pharmacokinetic monitoring during infusion was accomplished by dual silastic central venous catheters. The potential applications of the micropump infusion system as an alternative to current methods of continuous infusion in other species and in other studies are further discussed. This method provided a safe, reliable, and well-tolerated method of adjustable continuous intravenous infusion and simultaneous sampling of central venous blood in rabbits.     

Modulation of plasminogen activation and type IV collagenase activity by a synthetic peptide derived from the laminin A chain.

Laminin is a large multidomain glycoprotein with diverse biological activities which include stimulation of neurite outgrowth, enhancement of tumor metastasis, and promotion of cell growth, adhesion, and differentiation. A 19 amino acid synthetic peptide derived from the E8 fragment of the laminin A chain (Cys-Ser-Arg-Ala-Arg-Lys-Gln-Ala-Ala-Ser-Ile-Lys-Val-Ala-Val-Ser-Ala-Asp -Arg- NH2) was identified which promotes metastasis and stimulates collagenase IV activity in the culture medium of B16 melanoma cells (Kanemoto et al., 1990). We report that this peptide, here designated LamA2091-2108, is also a potent stimulator of tissue plasminogen activator (t-PA)-catalyzed plasminogen activation, resulting in a 22-fold increase in the kcat/Km of the activation reaction. The activity of purified type I and type IV collagenase was inhibited by LamA2091-2108 with IC50 values of 3 and 43 microM, respectively. These data support an alternative mechanism for the appearance of collagenase activity in the culture media of melanoma cells, namely, that the peptide stimulates plasminogen activation, subsequently generating collagenase activity.