Inhibition of thermolysin by bifunctional N-carboxyalkyl dipeptides.

A series of N-carboxyalkyl derivatives of L-leucyl-L-alanine was synthesized and tested as inhibitors of the zinc endoproteinase thermolysin. The purpose of the study was to determine whether bifunctional N-carboxyalkyl compounds with secondary metal coordinating groups are more potent inhibitors than analogs lacking such an additional binding function. Reductive condensation of L-leucyl-L-alanine (LA) with pyruvic, oxalacetic, alpha-ketoglutaric, 2-oxopentanoic, 4-ethyloxalacetic, or imidazoylpyruvic acids gave N-[1(R, S)-carboxyethyl]-LA (I), N-[1(R, S)-carboxy-2-carboxyethyl]-LA (II), N-[1(R, S)-carboxy-3-carboxypropyl]-LA (III), N-[1(R, S)-carboxy-n-butyl]-LA (IV), N-[1(R, S)-2-ethylcarboxyethyl]-LA (V), and N-[1(R, S)-carboxy-2-(4-imidazoyl-ethyl]-LA (VI), respectively. Values of KI determined with furylacryloyl-Gly-Leu-NH2 as substrate were 116 +/- 21, 7.4 +/- 1.8, 6.3 +/- 0.5, 19.7 +/- 1.5, 17.0 +/- 1.0, and 3.3 +/- 0.1 microM for compounds I-VI, respectively. Although bifunctional inhibitors II, III, and VI were indeed more potent than I, they were not much more effective than analogs IV and V that contained noncoordinating functionalities of comparable size. The results do not provide strong evidence for chelation of the active site zinc ion as proposed, although such interactions do not appear to be ruled out altogether.

Human recombinant transforming growth factor alpha stimulates bone resorption and inhibits formation in vitro.

Human recombinant transforming growth factor alpha (TGF alpha), which binds to the epidermal growth factor (EGF) receptor and causes several biological effects similar to those caused by EGF, was compared with murine EGF for its effects on a number of parameters of bone cell metabolism. TGF alpha stimulated bone resorption in two organ culture systems, the fetal rat long bone and neonatal mouse calvarial systems. TGF alpha stimulated bone resorption at concentrations as low as 0.1 ng/ml. TGF alpha effects on bone resorption in mouse calvariae were inhibited by indomethacin, suggesting that, like EGF, its effects were mediated by prostaglandin synthesis. TGF alpha had a different time course of action on bone resorption from that of EGF, causing more rapid release of previously incorporated 45Ca from bone cultures, suggesting that TGF alpha does not function on bone as a simple EGF analogue. TGF alpha also caused effects on osteoblast function resembling those of EGF. It inhibited alkaline phosphatase activity in cultured rat osteosarcoma cells with the osteoblast phenotype and inhibited collagen synthesis in fetal rat calvaria at concentrations of 1.0 ng/ml. The lowest concentration of TGF alpha (expressed as nanogram equivalents of EGF per ml) required to produce a response in all of the systems tested was about 1/10th of that needed for EGF to produce a similar effect. These results indicate that TGF alpha is a potent stimulator of bone resorption and inhibitor of bone formation as assessed by inhibition of collagen synthesis and alkaline phosphatase activity and are consistent with the hypothesis that TGF alpha may be responsible, at least in part, for the bone resorption associated with some tumors.

Schizophrenia as a semiotic disorder.

Lanin-Kettering and Harrow (1985) argue the traditional position that schizophrenia is a thought disorder. Chaika and Lambe (1985) counter that it is a speech disorder at the syntactic-discursive level, and not a thought disorder. On the basis of state-of-the-art research in linguistics, it is suggested that the symptoms of schizophrenia are evidence of neither a thought disorder nor a syntactic-discursive disorder but a semiotic disorder. Semiotic structures have the form of saying something about something to someone and involve speech act, reference, pragmatics, and interpretation. Therefore, it appears that schizophrenic disorder is located in this structure.

Identification of a messenger ribonucleic acid fraction in human prostatic cancer cells coding for a novel osteoblast-stimulating factor.

Prostatic cancer is frequently associated with new bone formation although the tumor-derived factors responsible for changes in bone cell function have not been identified. We have examined the synthesis of osteoblast-stimulating factors in a cultured human prostatic cancer cell line (PC-3) and show that conditioned medium from PC-3 cells stimulate mitogenesis and alkaline phosphatase in cells with the osteoblast phenotype (cultured rat osteosarcoma cells) and collagen synthesis in fetal rat calvaria. In order to characterize tumor-derived gene products which stimulate cells of the osteoblast phenotype messenger RNA (mRNA) was isolated from PC-3 cells and microinjected into Xenopus laevis oocytes. mRNA-directed translation products which were secreted into the oocyte medium were collected and assayed for a number of osteoblast stimulating properties. Translation products from PC-3 mRNA-injected oocytes stimulated division of cultured osteosarcoma cells by 8-fold and increased DNA synthesis as measured by incorporation of [3H]thymidine into these cells. In addition, tumor-derived translation products stimulated the production of alkaline phosphatase activity, a marker enzyme for bone formation, in cultured osteosarcoma cells. Oocytes injected either with water or with mRNA from a tumor not associated with bone formation were devoid of these activities. Total mRNA from the human prostatic cancer cells was then denatured and fractionated by size by agarose gel electrophoresis. When individual fractions of mRNA were eluted from the gel, translated in Xenopus oocytes, and the secreted translation products were tested for alkaline phosphatase-stimulating activity on osteoblast-like cells, the majority of the activity could be recovered in a mRNA fraction which was approximately 1800 bases in length. These results indicate that the PC-3 prostatic cancer cell line synthesizes a mRNA of approximately 1800 bases which codes for a heretofore unrecognized osteoblast-stimulating factor.

The bacterial content of breast milk after the early initiation of expression using a standard technique.

This study was initiated to evaluate the effect of early expression on the bacterial colony count of human milk. Significant bacterial contamination (greater than or equal to 10,000 colony-forming units/ml milk) was more common in 11 mothers who delayed the onset of expression of their milk compared with mothers who began to express their milk in the immediate postpartum period (n = 15) or who began to nurse their own full-term infants soon after delivery (n = 9). These data suggest that mothers who are separated from their prematurely born or sick infants should begin to express milk for their own infants as soon after birth as possible to provide milk with low bacterial contamination for frozen storage and later use.

An intercellular network artefact in glycol methacrylate dehydrated neonatal cartilage.

A striking network thought to consist of glycosaminoglycan in neonatal cartilage has recently been reported following glycol methacrylate (GMA) dehydration and embedding. This study attempted to learn why the network was not seen in other preparations previously reported. Comparative light and transmission electron microscopy of the network structures was carried out to determine their biological significance. We have shown the networks to be reproducible artefact, attained only following GMA dehydration of glutaraldehyde fixed neonatal cartilage.

The measurement of proteoglycan in the mineralizing region of the rat growth plate.

UNLABELLED: Using a recently developed technique for measuring proteoglycan by x-ray microprobe analysis, we examined six-week-old rat growth plates. In the proliferating zone, the proteoglycan concentration was found to be relatively low in the transverse septa, but somewhat higher in the longitudinal septa between the proliferating chondrocytes. In the lower hypertropic zone, in the region of the degenerating hypertrophic chondrocytes, the immediate pericellular area had a very high concentration of proteoglycan, as was obtained. These results indicate that either proteoglycan concentrations fall to very low levels in fully mineralized cartilage, or that the mineralization of cartilage interferes in some way with the measurement of microprobe signals from proteoglycans. We speculate that high levels of proteoglycan are necessary for mineralization in the epiphysis of the rat. CLINICAL RELEVANCE: The spot-to-spot analysis of proteoglycan by microprobe demonstrates the usefulness of this tool in analyzing minute volumes of cartilage for proteoglycan. It further demonstrates that mineralization of growth-plate cartilage is associated with changing patterns of concentration of proteoglycan. These normal patterns may constitute a basis for comparison in similarly evaluating concentrations of proteoglycan in diseases of growth disturbance.

Prospective clinical comparison of two methods for mechanical ventilation of neonates: rapid rate and short inspiratory time versus slow rate and long inspiratory time.

A prospective comparison was made of the clinical courses of two groups of neonates ventilated according to different protocols: one group at rates of 20 to 40/minute with a one-second IT, and the other at a rate of 60/minute and 0.5 second IT. Other ventilator settings were adjusted within protocol limits to maintain desired blood gas values. Mean starting and highest PIP were lower in the rapid rate group. The results showed no difference in mortality, failure to remain within protocol limits, time requiring respiratory treatment or FiO2 more than 0.6, and incidence of PDA or chronic lung disease. There was a difference (P = 0.011) in number of infants developing pneumothoraces (14% in the rapid group vs 35% in the slow group). Rapid rate ventilation can be used to decrease the incidence of pneumothorax, reserving long IT and higher PIP for infants who cannot be oxygenated or ventilated without them.

The use of brominated toluidine blue O in X-ray microanalysis for proteoglycan.

Toluidine blue O, a cationic dye has been used for visualizing proteoglycan by light and electron microscopy. Toluidine blue O has now been brominated and incorporated into epiphyseal cartilage during its fixation. Using energy dispersive X-ray microanalysis, bromine has been measured from area to area and compared to measurements of sulphur, a naturally occurring element of proteoglycan. Since bromine has been combined mole for mole with toluidine blue O and it is already known that toluidine blue O combines approximately mole for mole with the glycosaminoglycans of proteoglycan, the measurement of bromine, presents an indirect measure of proteoglycan concentration. The X-ray spectral localization of sulphur presents problems in X-ray analysis which are overcome with bromine which makes this technique attractive for workers in this field.