Effects of elution [correction of eution] conditions on the separation of calpastatin, mu- and m-calpain on DEAE-Sephacel chromatography.

A rapid stepwise measurement for the activities of calpastatin and mu- and m-calpains was developed by using 2-stage elution at pH 8.5 and then 7.0. The activities of calpastatin, mu-calpain and m-calpain can be rapidly assayed following the separation on DEAE-Sephacel chromatography by a 2 stage elution with 90 mM NaCl (pH 8.5), and then by 200 and 300 mM NaCl in elution buffer (pH 7.0). No significant differences in the recovery of these proteinases and inhibitor was observed between stepwise gradient and linear gradient methods.

Multigenerational effects of dietary fat carcinogenesis in mice.

The possibility of multigenerational transmission of a carcinogenic effect from exposure to a maternal diet high in fat was tested in mice. Diets with 2.6 or 29% fat (by weight) were fed to strain CD-1 mice during pregnancy. The female offspring were raised on a control diet (10% fat), mated, and continued on the control diet through pregnancy. Their female offspring were raised to terminal illness and autopsied. The total number of reproductive system tumors, pituitary tumors, and metastases was increased in the offspring with ancestral exposure to high dietary fat but to a lesser extent than had been reported previously for direct prenatal exposure to high maternal dietary fat. Because previous work has given evidence against germ cell transmission, a hypothesis based on a maternal effect was offered to explain the multigenerational carcinogenesis. These results have implications for epidemiological studies.

Effects of fostering on the increased tumor incidence produced by a maternal diet high in fat.

Previous work has shown that female offspring of mice fed a diet high in fat during pregnancy developed more reproductive system tumors and metastases than offspring of pregnant mice fed a low-fat diet. The purpose of the current experiment was to use fostering to test whether the sensitive period for this cancer effect involved the early postnatal period. Strain CD-1 female mice were placed on a diet of 2.6% fat or 29% fat from corn oil at 4 weeks of age and bred at 6-10 weeks of age. The special diets were discontinued at birth, and litters from dams that had been fed the low-fat diet were fostered to dams previously fed the high-fat diet, and vice versa. The offspring were raised to terminal illness and autopsied. There was no difference in age at terminal illness or in the number of the common nonreproductive system tumors between the two fostered groups. Tumor metastases appeared in both groups. However, the combined frequency of reproductive tract tumors and mammary tumors was significantly higher in mice exposed prenatally to a low-fat diet and fostered to dams that had consumed a high-fat diet during pregnancy than in mice exposed prenatally to a high-fat diet and fostered to a dam fed a low-fat diet. Thus the most sensitive period for a cancer effect from high fat was early postnatal, even though the special diets had been discontinued at birth. This matches the period of greatest sensitivity for sex differentiation of the hypothalamus.

Increased reproductive tract tumors in the female offspring of mice fed a high fat diet during the fetal stage of pregnancy.

In a previous study, female offspring of mice consuming a high fat diet throughout pregnancy developed reproductive system tumors and tumor metastases with a frequency significantly higher than offspring of mice on a low fat diet. To test for the sensitive period more specifically, the feeding of a high fat diet was restricted to the fetal period of pregnancy in the present experiment. The offspring were raised to terminal illness and autopsied. The total number of ovarian, uterine and mammary tumors was 14 among 74 mice exposed prenatally to low fat and 34 among 75 mice exposed prenatally to high fat (P < 0.002). In mice exposed to high fat during the fetal period 13 tumors produced metastases, but no metastases were identified after exposure to low fat (P < 0.001). Thus, a maternal diet high in fat during the fetal period of pregnancy was sufficient to increase reproductive system tumors and metastases in the female offspring.

Multi-generational carcinogenesis from diethylstilbestrol investigated by blastocyst transfers in mice.

Previous studies have shown that a carcinogenic effect can be transmitted from female mice exposed prenatally to diethylstilbestrol (DES) to their female offspring. Furthermore, male mice exposed pre-natally to DES can transmit a carcinogenic effect to their offspring through their germ cells. To study how multi-generational carcinogenesis is transmitted through females exposed pre-natally to DES, the technique of blastocyst transfer was utilized. Blastocysts from strain CD-1 mice exposed pre-natally to vehicle were transferred to mice exposed pre-natally to DES. Among 143 offspring from these transfers, there were 10 ovarian adenomas and 10 uterine adenocarcinomas. Among 92 offspring from blastocyst transfers between mice exposed pre-natally to vehicle only, there was 1 ovarian adenoma and 1 uterine adenocarcinoma. Thus the pre-natal exposure of the host to DES produced a maternal environment which increased the incidence of ovarian and uterine tumors. The reverse type of transfer was also performed, in which blastocysts from female mice exposed pre-natally to DES were transferred into mice exposed to vehicle only pre-natally. Among 99 offspring derived from DES-exposed germ cells, 6 developed ovarian adenomas and 16 developed uterine adenocarcinomas. Thus DES also has a multi-generational effect transmitted through the blastocyst, which is consistent with fetal germ cell mutation from DES.

Minifragmentation plating for comminuted distal patella fractures. A report of three cases.

A new technique using a minifragmentation plate to assist in the open reduction and internal fixation of displaced, comminuted distal-pole patella fractures was used in three patients with good results. This technique adds to the choices of treatment available to the orthopaedic surgeon who manages these difficult fractures.

Utilization of intracellular acylcarnitine pools by mononuclear phagocytes.

Carnitine is essential for the metabolism of long-chain fatty acids and has both direct and indirect roles in the metabolism of short-chain and medium-chain acyl-CoAs. The purpose of this study was to quantitate and identify the individual acylcarnitines that occur in human mononuclear phagocytes (MNP) after activating them with phorbol-12-myristate 13-acetate (PMA). Mononuclear phagocytes were isolated from healthy adults and the levels of free carnitine and individual acylcarnitines were determined in unactivated and activated cells. The degree of activation of MNP was assessed by following hydrogen peroxide production. In unactivated cells, acetyl-L-carnitine represented more than 80% of the total acylcarnitine pool. Small amounts of 3-carbon and 4-carbon acylcarnitines were present, with less than 10% of the carnitine pool being long-chain acylcarnitine. Free carnitine in unactivated cells represented 7% of the total carnitine pool, which remained essentially unchanged in unactivated cells when monitored for a period of 60 min. However, free carnitine rose to more than 50% of the total pool in PMA-activated cells. Similarly, after 1 h of activation, the acetylcarnitine level in activated cells decreased by more than 50%. These data suggest that acetylcarnitine plays a key metabolic role as MNP initiate an immune response. It was further shown that MNP contain both carnitine acetyltransferase and malonyl-CoA-sensitive carnitine palmitoyltransferase in mitochondrial-enriched fractions, as well as in post-mitochondrial supernatant fractions.

The molecular location of Ehrlich chromogen and pyridinoline cross-links in bovine perimysial collagen.

Collagenous peptides containing the Ehrlich chromogen (EC), a trifunctional cross-link of proposed pyrrolic structure, were selectively isolated from a tryptic digest of bovine perimysial collagen by coupling to a diazotised support. Peptides containing pyridinoline (Pyr), another trifunctional cross-link but based on a 3-hydroxypyridinium ring, were isolated from the uncoupled material. The isolated cross-linked peptides were purified by chromatographic procedures and subsequently characterised by amino acid and sequence analyses. EC occurred in stoichiometric amounts in three-chained peptides derived from type I collagen cross-link regions. In contrast, Pyr was found in non-stoichiometric amounts in three-chained peptides where two of the chains were identified as the 76 amino-terminal residues of the α1 (III) collagen chain. The third chain in these Pyr cross-linked peptides was derived from the C-terminal helical cross-link region of either type III collagen or the corresponding region of type I collagen, with the former region predominating. These findings suggest that EC and Pyr cross-links of perimysial collagen are associated mainly with type I and type III collagen respectively.

Pituitary tumors in mice exposed prenatally to diethylstilbestrol.

Hyperprolactinemia and prolactinomas are among the abnormalities reported for women exposed prenatally to diethylstilbestrol (DES). To pursue this issue in an animal model replicating the other abnormalities of prenatal DES exposure, pituitary glands were studied in the offspring of CD-1 mice receiving an i.p. injection of 1 or 2 micrograms DES/g body weight during late pregnancy. Among 132 mice exposed prenatally to DES and then raised to terminal illness, there were 24 pituitary tumors compared to only 1 tumor among 64 controls. The tumors consisted predominantly of cells with an eccentric nucleus and cytoplasm characterized by an acidophilic core and basophilic rim. These cells were identified as lactotrophs on the basis of prolactin immunohistochemistry and by an expected variation in frequency relative to physiological states. Evaluation of ovaries from the same mice revealed a deficiency of corpora lutea and an elevated incidence of ovarian tumors. These findings are consistent with abnormal sex differentiation of the fetal hypothalamus being the cause of most adverse effects from prenatal DES exposure.

Identification of the loci of the collagen-associated Ehrlich chromogen in type I collagen confirms its role as a trivalent cross-link.

Collagenous peptides containing the Ehrlich chromogen (EC) were selectively isolated from a tryptic digest of bovine tendon by coupling to a diazotized polyacrylamide support. The isolated p-phenol-azo-EC peptides were purified and characterized by amino acid and sequence analyses. EC occurred in stoichiometric amounts in trimeric cross-linked chains originating from the known cross-link regions of type-I collagen. The major locus of the EC was alpha 2(I)Hyl-933 x alpha 1(I)Lys(Hyl)-9N x alpha 2(I)Lys(Hyl)-5N but it was also shown to occur at the loci alpha 1(I)Hyl-87 x alpha 1(I)Lys(Hyl)-16C x alpha 1(I)Lys(Hyl)-16C and alpha 1(I)Hyl-930 x alpha 1(I)Lys(Hyl)-9N x alpha 2(I)Lys(Hyl)-5N. After sequence analyses of the C-terminal helical cross-link region alpha 2(I)928-963, corrections are presented for residues 927, 930, 932 and 933 of the bovine alpha 2(I) chain. The collagen-associated EC is postulated to be a trisubstituted pyrrole formed by the reaction of the aldehyde form of a telopeptidyl lysine residue with a bifunctional keto amino cross-link. It is also proposed that when the telopeptidyl lysine residue is hydroxylated the above reaction will result in pyridinoline formation.

Postoperative radial nerve paralysis caused by fracture callus.

This is a case of late-onset (6 weeks) radial nerve paralysis following open reduction and internal fixation of a comminuted distal humerus fracture. A transected radial nerve within the callus was found at reoperation. The mechanism of transection was thought to be repetitive motion of the nerve across an edge of new bone. The nerve was repaired and tendon transfers done subsequently with less than full functional recovery at 16 months. No such case has been previously reported.

The relationship between animal age and the thermal stability and cross-link content of collagen from five goat muscles.

The thermal stability of intramuscular collagen, as determined using differential scanning calorimetry, was measured in five muscles from 75 goats with known birth dates ranging in age from one day to 13 years. The collagen cross-link pyridinoline, and the collagen-associated, and putative cross-link, Ehrlich Chromogen were also measured. Five different muscles were examined and the effects of age compared to those found in the tendon of the longissimus dorsi muscle. The differences between intramuscular collagen and tendon collagen were found to be much greater than those between the intramuscular collagens of different muscles. Intramuscular collagen is more thermally stable than tendon collagen due to higher levels of heat-stable cross-links. However the increase in thermal stability of intramuscular collagen with age could not be explained simply in terms of the cross-links measured.

Collagen crosslinks and their relationship to the thermal properties of calf tendons.

The hydrothermal isometric tension and thermal transition temperature of collagen were determined in tendons from three different calf muscles. The levels of the nonreducible collagen crosslink, pyridinoline, and the collagen-associated Ehrlich chromogen were also measured in the three tendons. The reducible collagen crosslinks, hydroxylysinonorleucine, dihydroxylysinonorleucine, and histidinohydroxymerodesmosine were measured in two tendons. The thermal properties and levels of crosslinks were found to vary considerably between the different tendons, and also at different sites in two of the tendons. A strong correlation was observed between the thermal transition temperatures and the hydrothermal isometric tensions of the nine tendon sites examined. Both thermal properties correlated with the concentration of both pyridinoline and Ehrlich chromogen. The analogous behavior of the collagen-associated Ehrlich chromogen and the pyridinoline crosslink supports the role of the Ehrlich chromogen as a nonreducible crosslink.

Ipsilateral femoral neck and shaft fractures. Report of two cases using an alternate technique.

A technique for ipsilateral femoral neck and shaft fracture using the sliding compression hip screw with plate combined with trochanteric antegrade Ender nailing of the femur was applied in two cases. Ender nails can be passed without difficulty past a compression hip screw and the bicortical plating screws. The hip and femur can be fixed internally through a single approach in a single position. Sliding compression hip screw devices can provide excellent preliminary stable femoral neck fixation. Blood supply to the femoral head is not disturbed while the femoral intramedullary fixation is performed. Antegrade Ender nailing avoids the common knee complications associated with other retrograde techniques. Decreased operative time, less blood loss, less technical difficulty, and early mobilization are important factors in the multiple-injured patient. Femoral intramedullary fixation may require open reduction, circlerage to ensure stability, and maintenance of alignment in case of significant comminution to allow early crutch ambulation. This mode of fixation may be advantageous for selected cases.

Studies on the structure of connectin in muscle.

Enzymic hydrolysis, followed by amino acid analysis, provided no evidence for the presence of epsilon-(gamma-glutamyl) lysine or other isopeptide crosslinks in connectin. Gel elecrrophoresis in the presence of sodium dodecyl sulphate did not reveal any difference in connectin between normal and lathyritic muscle, indicating that lysyl oxidase does not initiate cross-link formation in connectin. Although connectin may be covalently crosslinked by some unknown mechanism, the available evidence suggests that the subunit of MW approximately to 900 000 is synthesised as a single polypeptide chain. In developing fetal muscle, myosin heavy chains are apparent some weeks earlier than connectin. This, together with the known susceptibility of connectin to hydrolysis, suggests that connectin exists in an exposed environment rather than as a core to the thick filament.

Proteolytic degradation of connectin, a high molecular weight myofibrillar protein, during heating of meat.

When homogenised muscle (pH 5·5) was heated at 55°C, connectin was extensively degraded, whereas actin and myosin heavy chains were apparently unaffected. It was concluded that carboxyl proteases (e.g. cathepsin D) were largely responsible, because the breakdown of connectin was inhibited by the addition of pepstatin. When whole muscle samples were heated at 50-70°C, degradation of connectin was inversely related to the ultimate pH of the source muscle, again indicating the role of carboxyl proteases. The greater activity of carboxyl proteases in tissues from older animals was apparently responsible for the more extensive degradation of connectin in muscle from older sheep. Because connectin is extensively degraded in cooked meat, it is unlikely to contribute to meat toughness.