Minimally invasive CAD/CAM lithium disilicate partial-coverage restorations show superior in-vitro fatigue performance than single crowns.

OBJECTIVE: To analyze the influence of restoration design (partial-coverage restoration vs. crown) and ceramic layer thickness on the performance and failure loads of CAD/CAM-fabricated lithium disilicate (LDS) reconstructions on molars after fatigue. MATERIALS AND METHODS: Seventy-two posterior monolithic CAD/CAM-fabricated LDS restorations (IPS e.max CAD, Ivoclar Vivadent) with different occlusal/buccal ceramic layer thicknesses (1.5/0.8, 1.0/0.6, and 0.5/0.4 mm) and restoration designs (PCR: non-retentive full-veneer/partial-coverage restoration, C: crown,) were investigated and divided into six groups (n = 12, test: PCR-1.5, PCR-1.0, PCR-0.5; control: C-1.5, C-1.0, C-0.5). LDS restorations were adhesively bonded (Variolink Esthetic DC, Ivoclar Vivadent) to dentin-analogue composite dies (Z100, 3M ESPE). All specimens were subjected to thermomechanical loading (1.2 million cycles, 49 N, 1.6 Hz, 5-55°C) and exposed to single load to failure testing. Failure analysis was performed with light and scanning electron microscopies. Data were statistically analyzed using ANOVA, Tukey-Test, and t-test (p < 0.05). RESULTS: Eight crown samples (C-0.5) and one PCR specimen (PCR-0.5) revealed cracks after fatigue, resulting in an overall success rate of 87.5% (crowns: 75%, PCRs: 96.88%). Direct comparisons of PCRs versus crowns for thicknesses of 0.5 mm (p < 0.001) and 1.0 mm (p = 0.004) were significant and in favor of PCRs. Minimally invasive PCRs (0.5 and 1.0 mm) outperformed crowns with the identical ceramic thickness. No difference was detected (p = 0.276) between thickness 1.5 mm PCRs and crowns. CONCLUSIONS: Minimally invasive monolithic CAD/CAM-fabricated posterior LDS PCRs (0.5 and 1.0 mm) resulted in superior failure load values compared to minimally invasive crowns. Minimally invasive crowns (0.5 mm) are prone to cracks after fatigue. CLINICAL SIGNIFICANCE: Minimally invasive CAD/CAM-fabricated LDS PCR restorations with a non-retentive preparation design should be considered over single crowns for molar rehabilitation.

[Skull base metastases with cranial nerve deficits : Clinical profile of a severe disease]. / Schädelbasismetastasen mit Hirnnervenaffektionen : Klinischer Steckbrief einer schwerwiegenden Erkrankung.

BACKGROUND AND PURPOSE: Skull base metastases are a severe complication of various malignant tumors. If cranial nerves are involved even small lesions can cause significant symptoms. Specific clinical characteristics like neurological symptoms, associated primary tumors, prognosis and optimal treatment are poorly defined and are systematically described in this article. METHODS: In a monocentric retrospective study patients with skull base metastases and cranial nerve deficits who received treatment between 2006 and 2018 were analyzed concerning clinical characteristics at initial diagnosis, treatment and course of the disease. RESULTS: In this study 45 patients with skull base metastases and cranial nerve deficits were included. The most frequent primary tumors were prostate cancer (27%), breast cancer (22%) and multiple myeloma (16%). The most involved cranial nerves were trigeminal nerve (42%), oculomomotor nerve (33%) and facial nerve (27%). Of the patients 84% had additional bone metastases outside the skull base. Dural infiltration or meningeal carcinomatosis were each observed in 13% of the patients. After radiotherapy cranial nerve deficits remained stable in 61% of all cases and in 22% symptoms improved. Median overall survival from treatment was 8 months (range 0.4-51 months). Patients with dose-escalated radiotherapy appeared to live longer (16.4 months vs. 4.7 months). This effect persisted in a multivariate analysis including the Karnofsky index, number of metastases, primary tumor and radiation dose (HR 0.37, p = 0.02). CONCLUSION: Skull base metastases with cranial nerve deficits are complex diseases with poor prognosis. Precise diagnosis and treatment are required. Further research is needed to improve treatment.

Relationship between milk constituents from milk testing and health, feeding, and metabolic data of dairy cows.

A new evaluation scheme to assess the nutritional status of dairy cows on the basis of milk constituents was derived from 7.37 million German records of milk testing. The aim of this work was to validate this new scheme. Two data sets with fertility and health information (data set A) and with measured energy and nutrient intake and metabolic characteristics (data set B) were analyzed. Data set A included 32 commercial dairy farms in northeast Germany, with 72,982 records of 43,863 German Holstein cows; data set B included 12 German experimental farms, with 49,275 records of 1,650 German Holstein, Simmental, and Brown Swiss cows. Milk traits were linked to health disorders and metabolic and feeding characteristics. Frequently used limits of milk constituents were compared with ranges of the new "Dummerstorf feeding evaluation." To distinguish an optimal from a deficient energy supply, a milk protein content ≥3.20% (previously used) and a milk fat:protein ratio (FPR) ≤1.4 (new scheme) were chosen and compared with feed energy intake in relation to demand. Energy status was more often correctly assigned by FPR than by milk protein content (80.7 and 68.7%, respectively). Over all data, the new optimum range of milk urea between 150 and 250 mg/L was better suited to dietary crude protein intake in relation to demand than the previously used range of 150 to 300 mg/L (42.4 and 38.0%, respectively). Ketosis or blood values associated with ketosis such as ß-hydroxybutyrate >1.2 mmol/L or nonesterified fatty acids >1,000 µmol/L, as well as strong mobilization of body weight ≥1.5 kg/d, loss of back fat thickness ≥10 mm, and loss of body condition score ≥1 unit in first 60 days in milk were compared with different milk trait thresholds. For the updated scheme FPR >1.4 was used in combination with either milk protein content below the individual statistical lower limit of milk protein content, or milk fat content greater than the individual statistical upper limit of milk fat content; FPR >1.5 was taken as a frequently used threshold. For these ketosis indicators, the new scheme had higher sensitivities. Energy oversupply or the risk of overconditioning could not be identified by milk constituents alone. Urinary acid-base content was not related to milk content. Similarly, milk testing data did not allow a clear distinction to be made between the diagnoses of acidosis and, for example, ketosis. Essential requirements for good herd management are the continuous observation of milk testing data in combination with other established instruments of feeding and animal monitoring.

Establishment of a Mucin Secreting Cell Line Cx-03 from an Uterine Carcino Sarcoma.

PURPOSE: The identification of novel cell lines which combine the most important properties of mucosal membranes in terms of drug absorption, transmembrane transport and mucus secretion can help to establish improved and meaningful test systems for pharmacological and infectiological studies. METHODS: We have established a novel mucus secreting tumor cell line (Cx-03) derived from a female patient who underwent radical hysterectomy after diagnosis of a large malignant carcino sarcoma (Muellerian mixed tumor). Via xenotransplantation in SCID beige mice, recultivation and subcloning a stable cell line was established from primary tumor cells. RESULTS: Human origin and novelty of the cell line was determined by karyotype analysis and STR fingerprint. During growth cells produce considerable amounts of a PAS positive viscoelastic mucus. Immunostaining revealed expression of mucins and the mucin modifier CLCA1. We demonstrate in initial electrophysiological experiments that confluent, polarized monolayers of Cx-03 are formed (on PCF-filter supports) that exhibit stable electrical resistance (> 600 Ω cm2). Confluent Cx-03 monolayers express barrier-forming tight junction proteins claudin-1 and -4 which co-localize with zonula occludens protein-1 (ZO-1) at cell-cell contacts. CONCLUSIONS: Mucus secretion is a rare property among mammalian cell lines. In combination with its ability to form polarized monolayers Cx-03 might contribute as a novel cell based model for drug absorption, transport and barrier studies.

Bubbles in live-stranded dolphins.

Bubbles in supersaturated tissues and blood occur in beaked whales stranded near sonar exercises, and post-mortem in dolphins bycaught at depth and then hauled to the surface. To evaluate live dolphins for bubbles, liver, kidneys, eyes and blubber-muscle interface of live-stranded and capture-release dolphins were scanned with B-mode ultrasound. Gas was identified in kidneys of 21 of 22 live-stranded dolphins and in the hepatic portal vasculature of 2 of 22. Nine then died or were euthanized and bubble presence corroborated by computer tomography and necropsy, 13 were released of which all but two did not re-strand. Bubbles were not detected in 20 live wild dolphins examined during health assessments in shallow water. Off-gassing of supersaturated blood and tissues was the most probable origin for the gas bubbles. In contrast to marine mammals repeatedly diving in the wild, stranded animals are unable to recompress by diving, and thus may retain bubbles. Since the majority of beached dolphins released did not re-strand it also suggests that minor bubble formation is tolerated and will not lead to clinically significant decompression sickness.

Belowground inter-ramet water transport capacity in Populus euphratica, a Central Asian desert phreatophyte.

Populus euphratica Oliv. is a widespread phreatophytic tree species that forms riparian forests in (hyper-)arid regions of Central Asia. Its recruitment strongly relies on vegetative propagation from 'root suckers' that emerge from underground root spacers. The water transport through the spacers, although decisive for emerging ramets, has only rarely been quantified, but is crucial for the vegetative regeneration of the forests. In root spacers with different diameters collected from a mature poplar forest in northwest China, we calculated the hydraulic conductivity (kc ) from anatomical investigations on the basis of a modified Hagen-Poiseuille equation and measured it (km ) with a perfusion solution in the laboratory. The km values were compared with the water use by young and mature P. euphratica trees determined in previous studies. We obtained a significant correlation between km and kc (which, however, was higher by at least one order of magnitude). Due to the extensive occurrence of tyloses, particularly in older conduits and thicker spacers, and because the conduit area did not increase with spacer diameter, neither kc nor km increased with an increase in spacer diameter. The water supply through the spacers would be sufficient to meet the water demand even of mature trees. Our results provide a mechanistic explanation for the observed occurrence of P. euphratica clones across large areas and, provided that they are also valid for stands with larger distances to the water table, for the sustained growth and vegetative reproduction of P. euphratica stands growing at larger distances from the groundwater.

Neurotrophin-3, but not nerve growth factor, promotes survival of human intestinal mast cells.

Neurotrophins are potent regulators of neuronal cell survival and function. Nerve growth factor (NGF) was shown to reduce apoptosis in cord blood-derived mast cells. Here, we examined the effect of the neurotrophins NGF and neurotrophin (NT)-3 on survival and mediator release of human intestinal mast cells. Mast cells isolated from normal intestinal tissue were cultured in the presence of NGF, NT-3, or stem cell factor (SCF) alone or in the presence of SCF together with each neurotrophin. NGF or NT-3 alone did not promote mast cell survival. In contrast, mast cell recovery was increased twofold when mast cells were cultured with NT-3 in addition to SCF for 14 days compared with control. Mast cell recovery was further increased following a combined addition of NT-3, SCF and IL-4. NT-3 mediated mast cell growth was dependent on the primary receptor for NT-3 TrkC. NGF in combination with SCF or with SCF and IL-4 showed no effect on mast cell survival. Histamine release and histamine content per mast cell remained unchanged, whereas leukotriene C4 release decreased if mast cells were cultured with NGF or NT-3 in addition to SCF. In summary, NT-3 affects mature human mast cells by promoting mast cell survival, whereas NGF does not.

Complete failure of spinal anaesthesia in obstetrics.

Intrathecal anaesthesia, either as a single shot-spinal or as part of a combined spinal-epidural technique, is now widely accepted as the management of choice for caesarean section. It generally produces rapid and predictable anaesthesia, yet occasionally fails for no apparent reason. Four case reports of seemingly inexplicable complete failure of intrathecal anaesthesia are presented, together with a literature review of other cases and possible causes of the failure, which include anatomical abnormality, drug failure and management failure.

4'-Chlorodiazepam is neuroprotective against amyloid-beta in organotypic hippocampal cultures.

The translocator protein (TSPO) is an outer mitochondrial membrane protein involved in the transport of cholesterol into the mitochondria, which is the first step for the synthesis of steroid hormones, as well as in the regulation of mitochondrial permeability transition pore opening and apoptosis. Studies have shown that the activation of TSPO may promote neuroprotective actions in experimental models of neurodegeneration and brain injury. In a previous study, our group showed that 4'-chlorodiazepam (4'-CD), a TSPO ligand, was neuroprotective against amyloid-beta (Aß) in SHSY-5Y neuroblastoma cells. The aim of this study was to evaluate if 4'-CD was also neuroprotective against Aß in organotypic hippocampal cultures and to identify its mechanisms of action. Aß decreased the cell viability of organotypic hippocampal cultures, while 4'-CD had a neuroprotective effect when administered at 100nM and 1000nM. The neuroprotective effects of 4'-CD against Aß were associated with an increased expression of superoxide dismutase (SOD). No differences were found in the expression of catalase, glial fibrillary acidic protein, Akt and procaspase-3. In summary, our results show that 4'-CD is neuroprotective against Aß by a mechanism involving the modulation of SOD protein expression.

Autocrine tumor cell growth-inhibiting activities from human malignant melanoma.

Autocrine-secreted tumor cell growth-inhibiting activities were isolated from supernatants of a malignant melanoma cell line, HTZ 19-dM, established from a central nervous system melanoma metastasis. HTZ 19-dM was characterized by cyto- and immunocytochemistry and karyotyping; cells were propagated in defined serum-free tissue culture medium for up to 8 months. Supernatants were ultrafiltrated, dialyzed, lyophilized, and purified by Bio-Gel P-10 gel permeation chromatography, leading to three active fraction pools, MIAI [melanoma-inhibiting activity (MIA), 2 kDa), MIAII (Mr 11,500-17,000) and MIAIII (proteins at the cutoff of Bio-Gel P-10) inhibiting growth of 19-dM cells with 50% inhibitory concentrations of 0.79 microgram/ml (MIAI), 0.13 microgram/ml (MIAII), and 16.7 micrograms/ml (MIAII). MIAII could be further purified by reverse phase high pressure liquid chromatography; the main activity displayed a 50% inhibitory concentration of 0.33 microgram/ml. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis one major band (molecular weight about 14,000) and two minor bands (up to Mr 17,000) were identified. Macromolecular synthesis was inhibited in 19-dM cells up to greater than 99.5%; tumor stem cell colony formation was reduced by 99.89%; the inhibitory effect of MIAII was irreversible, nonsaturable, and partially antagonized by a serum factor (depending on purification stage). MIAII was heat stable (3 min at 100 degrees C) and trypsin labile. The effect of MIAII on allogeneic neuroectodermal tumors was also investigated; proliferation of two of three malignant melanomas and two of four glioblastomas was inhibited up to 85.2%; proliferation of a neuroblastoma cell line could be inhibited to 33.8%, whereas normal fibroblasts and low grade gliomas were not influenced in their proliferation.

Formation of noncanonical high molecular weight caspase-3 and -6 complexes and activation of caspase-12 during serum starvation induced apoptosis in AKR-2B mouse fibroblasts.

Apoptosis is mainly brought about by the activation of caspases, a protease family with unique substrate selectivity. In mammals, different complexes like the DISC complex or the apoptosome complexes have been delineated leading to the cleavage and thus activation of the executioner caspases. Although caspase-3 is the main executioner caspase in apoptosis induced by serum starvation in AKR-2B fibroblasts as demonstrated by affinity labeling with YVK(-bio)D.aomk and partial purification of cytosolic extracts by high performance ion exchange chromatography, its activation is apparently caused by a noncanonical pathway: (1) Expression of CrmA, an inhibitor of caspase-8, failed to suppress apoptosis; (2) There was no formation of high molecular weight complexes of Apaf-1 indicative for its activation. Furthermore no cleavage of caspase-9 was observed. But surprisingly, gelfiltration experiments revealed the distribution of caspase-3 and -6 into differently sized high molecular weight complexes during apoptosis. Though the apparent molecular weights of the complexes containing caspase-3 (600 kD for apoptosome and 250 kD for microapoptosome) are in accordance with recently published data, the activity profiles differ strikingly. In AKR-2B cells caspase-3 is mainly recovered as uncomplexed enzyme and in much lower levels in the apoptosomes. Remarkably, the 600 kD and 250 kD complexes containing activated caspase-3 were devoid of Apaf-1 and cytochrome c. In addition a new 450 kD complex containing activated caspase-6 was found that is clearly separated from the caspase-3 containing complexes. Furthermore, we disclose for the first time the activation of caspase-12 in response to serum starvation. Activated caspase-12 is detectable as non-complexed free enzyme in the cytosol.

ATP and adenosine prevent via different pathways the activation of caspases in apoptotic AKR-2B fibroblasts.

Confluent AKR-2B fibroblasts rapidly disintegrate after serum deprivation.27 ATP or adenosine added immediately after serum removal afforded substantial protection against cell death even for a long period of 24 h. ED50 values were 14 and 110 microM for ATP and adenosine, respectively. In the presence of 5 microg/ml cycloheximide the protective effect of both substances was suppressed, indicating that protein synthesis is required. The protective effect of ATP was highly specific since among numerous tested derivatives only ATP-[gamma-S] exhibited a substantial protective effect. The ability of ATP and adenosine to modulate cell division was analyzed. Both substances did not exhibit any mitogenic effect. Adenosine completely blocked PDGF-BB induced cell division, whereas ATP had no effect. Unlike adenosine, ATP strongly stimulated Ca2+-release from intracellular stores. On the other hand, adenosine stimulated an increase in the intracellular concentration of cAMP from 0.4 - 1.5 microM, whereas ATP decreased the content below 0.1 microM. ATP stimulated the phosphorylation of MAP-kinase, RSK and p70S6-kinase; adenosine was inactive. After complexation of [Ca2+]i the protective effect of ATP was greatly lost while adenosine was still active. Surprisingly neither ATP nor adenosine caused an activation of PKC-isoforms. After incubation with pertussis toxin, the protection by ATP was reduced indicating an involvement of Gi-proteins in the signal transduction induced by ATP. Our results indicate that ATP as well as adenosine are potent inhibitors of cell death caused by serum deprivation and that this protective effect apparently occurs via distinct pathways. However, both pathways must converge at the point of caspase activation, since the stimulation of DEVDase- and VEIDase-activities, respectively, are suppressed by either ATP or adenosine.

Regulation of Gadd45a mRNA expression in vascular smooth muscle under growth and stress conditions.

In order to identify differentially expressed genes under growth conditions, quiescent vascular smooth muscle cells (VSMCs) were stimulated with foetal calf serum (FCS) or platelet-derived growth factor-BB (PDGF-BB) for different time periods. Analysing the gene expression by the differential display (DD) method, we identified the cDNA of the growth arrest and DNA damage inducible gene 45a (Gadd45a, also known as gadd45 and gadd45a). Treatment with FCS or PDGF-BB led to a transient down-regulating of Gadd45a expression during the G0/G1 phase and maximal expression when cells had completed division. We found that expression of p53 and BRCA1 mRNA precedes Gadd45a mRNA expression with a maximal induction in the S phase. As in smooth muscle cells, a similar pattern of the Gadd45a mRNA expression was observed in knockout Gadd45a(-/-) cultured mouse embryonic fibroblasts (MEFs). However, no differences between Gadd45a(+/+) and Gadd45a(-/-) cell lines were observed regarding their kinetics of cell division. These experiments suggest a function of Gadd45a when cells exit the cell cycle rather than when regulating the entry into the S phase.

Down-regulation of Akt/PKB in senescent cardiac fibroblasts impairs PDGF-induced cell proliferation.

OBJECTIVE: Cardiovascular diseases are the leading cause of death in the Western World, especially in the elder population. One pathophysiological component of cardiovascular disease is myocardial fibrosis, primarily derived from cardiac fibroblasts. Here we investigated the regulation of proliferation of fibroblasts from hearts of adult rats by platelet derived growth factor AA (PDGF-AA). METHODS: Cardiac fibroblasts were isolated from adult Wistar rats. PDGF-induced cell proliferation was analysed by FACS. PDGF-receptor numbers were analysed by receptor binding assays. Using differential display, differentially expressed kinases were identified during ageing in vitro and confirmed by Northern and Western blotting. Transient overexpression of IRES-GFP constructs was used to analyse the role of the akt kinase on proliferation by FACS. RESULTS: During in vitro senescence/aging of primary fibroblasts, the growth response to PDGF-AA was greatly reduced without alterations in its receptor number or affinity and without changes in downstream signalling via the MAP-kinase pathway. By using a differential display strategy selective for protein kinases, we identified reduced expression of Akt-1 kinase (PKB-alpha) in senescent rat cardiac fibroblasts. These findings were supported by data showing reduced expression of Akt-1 in heart samples from old humans. Overexpression of activated Akt-1 almost completely reconstituted PDGF-AA dependent cell proliferation in aged fibroblasts. CONCLUSION: These results support an important role for Akt in senescence and regulation of cardiac fibroblast cell proliferation.

Effect of PDGF-AB heterodimer on a corneal epithelial cell line.

Exposure of cells of a rabbit corneal epithelial cell line (SIRC) to platelet-derived growth factor-AB heterodimer (PDGF-AB) resulted in a rapid and transient elevation of cytosolic free calcium concentration with a maximum at 2 to 3 min after stimulation. The kinetics of the calcium response were dose-dependent, e.g., higher concentrations of PDGF-AB caused a faster rise in cytosolic free calcium concentration. Maximum response was achieved with 10 ng/ml PDGF; higher concentrations up to 100 ng/ml did not further enhance cytosolic free calcium concentration. The ED50 was calculated to be 5 ng/ml PDGF-AB. After complexing extracellular calcium, PDGF-AB still caused a significant rise in cytosolic free calcium concentration indicating a mobilization of calcium from intracellular stores. This rise, however, was less pronounced than in the presence of extracellular calcium. The elevation in cytosolic free calcium concentration was not accompanied by an increased mitotic or proliferative activity of the cells as checked by [3H]thymidine incorporation and counting of cell numbers after 3 days of continuous incubation with various concentrations of PDGF-AB or by alterations in cell size and cell volume. In contrast, alterations in cell shape with a remarkable amount of rounded and partially detached SIRC cells after addition of PDGF-AB were observed within 24 h. Moreover, PDGF-AB caused a reversible distortion of cytoskeletal components such as actin-containing microfilament bundles, microtubules, and vimentin filaments. The results suggest that PDGF-AB may act only as a competence factor for the stimulation of SIRC cells via modification of the intracellular calcium homeostasis.

Gangliosides GM1, GM2 and GM3 inhibit the platelet-derived growth factor-induced signalling transduction pathway in vascular smooth muscle cells by different mechanisms.

Gangliosides appear to regulate proliferation of different cell types. In the present study, we investigated the effects of gangliosides GM1, GM2 and GM3 on platelet-derived growth factor (PDGF)-induced vascular smooth muscle cell (VSMC) growth. In addition, we examined the effects of gangliosides on the PDGF-BB-dependent signalling transduction pathway in rat aortic VSMC. GM2 and GM1 inhibit the PDGF-BB-dependent receptor tyrosine autophosphorylation, stimulation of the PLC-gamma 1, increase of inositol-1,4,5-trisphosphate (InsP3), elevation in cytosolic free Ca2+ ([Ca2+]i), expression of the immediate early growth response gene c-fos and cell proliferation with the following rank order of potency GM2 > GM1. Although GM3 did not influence the PDGF-BB-dependent receptor autophosphorylation and PLC-gamma 1 activation, it effectively inhibited the PDGF-BB-dependent InsP3 formation, [Ca2+]i and cell growth. Binding studies with 125I-PDGF-BB on VSMC in the presence and absence of 10 to 50 microM of each ganglioside revealed that GM1 and GM2 effectively inhibited the specific binding of PDGF-BB with an IC50 value of 20 microM for GM2 and 30 microM for GM1. GM3 had no significant effect on the specific 125I-PDGF-BB binding. These observations suggest that GM1 and GM2 may interact with PDGF-BB or its receptor resulting in a prevention of its binding. GM3 was able to suppress the PDGF-BB-dependent increase of InsP3 and [Ca2+]i downstream of the PDGF-BB-dependent receptor autophosphorylation and PLC-gamma 1 activity.

Differential effects of platelet-derived growth factor isoforms on plasminogen activator activity in fetal rat osteoblasts due to isoform-specific receptor functions.

We examined receptor binding and metabolic effects of the platelet-derived growth factor (PDGF) isoforms AA, AB, and BB in cultures of osteoblastic cells from fetal rat calvaria. Saturation binding experiments demonstrated the presence of 6,000 binding sites for PDGF-AA, 42,000 for PDGF-AB, and 60,000 for PDGF-BB. Binding competition experiments were compatible with the recently postulated model of three PDGF receptor subtypes with differential affinity for the PDGF isoforms. The effects of the PDGF isoforms on DNA synthesis, collagen synthesis, and alkaline phosphatase activity in osteoblasts strictly correlated with the number of available binding sites. Accordingly, PDGF-BB was the most potent isoform, PDGF-AB was slightly less potent, and PDGF-AA was the least potent. In contrast, we found that PDGF-BB was less potent than PDGF-AB in increasing plasminogen activator activity in the osteoblast-conditioned medium. Our data strongly suggest that the PDGF receptor subtypes in fetal rat osteoblasts not only selectively bind one or more PDGF isoforms, but are also capable of responding differently to these isoforms.

Cloning, sequencing and expression in Escherichia coli of the D-2-hydroxyisocaproate dehydrogenase gene of Lactobacillus casei.

D-2-Hydroxyisocaproic acid dehydrogenase (D-HicDH) from Lactobacillus casei was purified and partially sequenced. A 65-mer oligodeoxyribonucleotide probe corresponding to the N-terminal 23 amino acids was synthesized and a physical map was made of the genomic region which hybridized most strongly. A strongly hybridising restriction fragment was highly purified and eventually cloned at low frequency in pBR322. The original clones spontaneously produced D-HicDH at about 0.05% of total protein and showed viability problems in that 10- to 12-h growth-lag periods occurred after diluting stationary cultures into fresh medium. Subcloning into pGEM3 plasmids for sequencing with concomitant ExoIII deletion led to clones which no longer exhibited the growth inhibition characteristics but now made D-HicDH as 3 to 5% of total protein. Subcloning downstream from a double PL PR promoter in plasmid pJLA601 gave a highly inducible clone that builds large inclusion bodies of largely denatured D-HicDH. The gene transcript was mapped for L. casei and Escherichia coli hosts. The promoter, terminator and Shine-Dalgarno sequence are functional in both organisms. The gene encodes a protein subunit of 38 kDa, whereby 67% of the sequence could be checked by correlation with partial peptide sequences from the original enzyme. So far no Lactobacillus gene has been found to utilize the Arg codons AGG and AGA.

Identification of individual amino acids in platelet-derived growth factor that contribute to the specificity towards the beta-type receptor.

Platelet-derived growth factor constitutes a family of three isoforms (PDGF-AA, -AB, and -BB) composed of two homologous polypeptide chains (A and B). These isoforms interact with two types of receptors termed alpha or beta. Whereas PDGF-AA binds only to the alpha-receptor, PDGF-BB binds and activates both receptors with high affinity. To map regions that are specific for the beta-receptor, we introduced mutations into PDGF-AA located in previously identified epitopes [1991, Biochemistry 30, 3303-3309]. A single amino acid exchange in domain II of PDGF-AA (Ala67----Arg) was sufficient to bring about a reduced but significant activation of the beta-receptor. In domain I the exchange of residues Pro26----Arg together with Ser28----Asn switched the specificity towards the beta-receptor. These data indicate that parts of the exposed domains are indeed involved in receptor binding. Since these single mutations lead to mutant proteins which are about 100-fold less active than PDGF-BB, it is suggested that other amino acid residues also participate in the binding to the receptors.